Leukemias are cancers of white blood cells. The cells grow and divide rapidly, often because of mutations in proteins called kinases. Since the kinase mutations do not occur in healthy cells, they provide a good target for anti-leukemia drugs. Several such kinase inhibitors are effective at treating leukemia patients. However, most leukemia cells develop ways to resist the effects of the kinase inhibitors over time, leading to relapses of the disease.

One way that leukemia cells resist kinase inhibitors is by taking advantage of signals coming from supportive cells, known as stromal cells, in the bone marrow. When patients are treated with kinase inhibitors, the bone marrow stromal cells produce more of a signaling protein called FGF2. The leukemia cells then use FGF2 to survive the effects of the kinase inhibitors.

It was not clear how the FGF2 signal reaches the leukemia cells from the bone marrow stromal cells. Now, using biochemical techniques, Javidi-Sharifi, Martinez et al. show that bone marrow stromal cells package FGF2 into small compartments called exosomes. The stromal cells release the exosomes into the bone marrow, and the leukemia cells then engulf and internalize the exosomes. Leukemia cells that had taken up FGF2 in this way were better able to survive kinase inhibitor treatment than leukemia cells that had not.

Javidi-Sharifi, Martinez et al. also observed that FGF2 also affects the bone marrow stromal cells themselves, causing them to grow faster, produce more FGF2 and release more exosomes. Blocking the effects of FGF2 on the stromal cells slowed their growth and caused fewer exosomes to be released. In addition, mice whose bone marrow stromal cells could not produce FGF2 survived leukemia for longer than mice whose stromal cells provided protective FGF2 in exosomes to leukemia cells. This suggests that taking advantage of drugs that prevent bone marrow stromal cells from releasing FGF2 in exosomes might improve treatments for leukemia. Further research will be needed to confirm whether this strategy would be effective in humans.

TKIs have revolutionized the treatment of CML and have shown promise in AML, however development of resistance remains a problem. In CML, resistance develops in a minority of patients, and is most often caused by resistance mutations. However, some patients still develop resistance in the absence of known resistance mutations. In contrast, development of resistance in AML is the norm. Inhibitors of mutated FLT3, which is present in about 30% of AML patients, are initially quite efficacious (Smith et al., 2012). However, resistance to FLT3 kinase inhibitors in AML typically develops within a few months. In some cases, resistance is cell-intrinsic and due to secondary mutations in the activating loop of FLT3 that prevent drug binding (Weisberg et al., 2009), however, resistance still develops in the absence of these mutations. Within the marrow microenvironment, leukemia cell survival can be mediated by extrinsic ligands that activate alternative survival pathways (Smith et al., 2017; Ghiaur and Levis, 2017; Wilson et al., 2012) and over time can lead to development of intrinsic resistance mutations (Wilson et al., 2012; Traer et al., 2012).

Bone marrow stromal cells provide a supportive structure and secrete cytokines that contribute to the normal hematopoietic stem cell niche, but can also protect leukemic cells from therapy (Colmone et al., 2008; Ayala et al., 2009). Initial studies into the mechanisms of resistance utilized normal marrow stroma (Manshouri et al., 2011), but the stroma can be altered by leukemia, in a manner similar to development of cancer associated fibroblasts in solid tumors (Paggetti et al., 2015; Huang et al., 2015; Huan et al., 2015). We found that fibroblast growth factor 2 (FGF2) expression is increased in marrow stromal cells during tyrosine kinase inhibitor (TKI) therapy and protects leukemia cells (Ware et al., 2013; Traer et al., 2014; Javidi-Sharifi et al., 2015; Traer et al., 2016). FGF2 has also been shown to be essential for stress hematopoiesis after chemotherapy (Itkin et al., 2012; Zhao et al., 2012), suggesting that leukemia cells can hijack a normal marrow stress response for their own survival.

Despite its important roles in physiology and pathology, several aspects of FGF2 biology remain poorly understood. FGF2 does not have a signal peptide and is not secreted through the canonical secretory pathway. Alternative mechanisms for secretion have been proposed, but how FGF2 is conveyed between two cells remains unclear (Steringer et al., 2015; Zacherl et al., 2015). Additionally, while recombinant FGF2 directly stimulates myeloid colony formation (Berardi et al., 1995), there are also reports suggesting that FGF2 can indirectly regulate hematopoiesis by stimulating stromal cells to produce cytokines (Avraham et al., 1994).

We discovered that FGF2 is largely secreted in extracellular vesicles (ECVs) and exosomes from bone marrow stromal cells. ECVs are able to protect leukemia cells from the effects of TKI therapy. Furthermore, autocrine FGF2-FGFR1 activation in marrow stromal cells increases the secretion of FGF2-laden exosomes, indicating that exosome secretion is regulated in part by FGF2-FGFR1 signaling. Inhibition of FGFR1 can reverse this protective stroma-leukemia interaction and restore leukemia cell TKI sensitivity in the marrow niche.

The normal hematopoietic microenvironment is altered by leukemia, and can protect leukemia cells from the effects of both chemotherapy and targeted kinase inhibitors (Yang et al., 2014; Parmar et al., 2011; Manshouri et al., 2011; Traer et al., 2014; Traer et al., 2016). Until recently, stromal protection of leukemia cells was thought to be largely mediated by secreted cytokines or through direct contact (review [Meads et al., 2009]). Here, we show that exosomes from bone marrow stromal cells are transferred to leukemia cells, and protect them from kinase inhibitors. Exosomes have previously been identified as important mediators of malignancy, including recent reports of leukemia exosomes modulating marrow stroma (Paggetti et al., 2015; Huan et al., 2015; Peinado et al., 2012; Filipazzi et al., 2012). We found that the reciprocal transfer also occurs, and that marrow stromal exosomes efficiently protect leukemia cells from targeted kinase inhibitors. Along with recent reports that entire mitochondria are transferred between stromal cells and leukemia cells during therapy (Marlein et al., 2017; Moschoi et al., 2016), our data adds to an increasingly complex and intimate relationship between marrow stromal cells and leukemia cells. Indeed, it is almost hard to imagine the leukemia cell in the niche as a separate entity given the direct exchange of organelles, ECVs, cell-cell signaling, and secreted cytokine signaling between stromal and leukemia cells. A better understanding of this relationship is important to develop better ways to eradicate leukemia cells and cure more patients.

Isolated reports have previously suggested that FGF2 is contained in ECVs (Proia et al., 2008; Choi et al., 2016), but FGF2 has also been reported to self-assemble into a pore-like structure on the cell membrane and mediate its own translocation with the help of extracellular heparan sulfate (Steringer et al., 2015; Schäfer et al., 2004). Compared to other soluble secreted cytokines, FGF2 was uniquely enriched in ECVs and exosomes (Figure 2), suggesting that secretion in ECVs is the primary mechanism of FGF2 paracrine signaling from marrow stromal cells. Since FGFR1 is also found on exosomes (Figure 3D), the FGF2-FGFR1 interaction on exosomes may play a direct role in loading FGF2 in exosomes and/or regulate secretion. FGFR inhibitors also increase the amount of FGFR1 protein in stromal cells as measured by immunoblot, consistent with a role in receptor cycling and/or reduced secretion in exosomes.

Similar to our observations, epidermal growth factor receptor has been shown to be secreted on ECVs, and secretion is increased after ligand stimulation (Sanderson et al., 2008; Perez-Torres et al., 2008). Likewise, overexpression of oncogenic HER2 in breast cancer cell lines resulted in qualitative differences in microvesicle content (Amorim et al., 2014), suggesting a role for activated receptor tyrosine kinases in exosome production and secretion. Receptor-mediated endocytosis is the first step of exosome biogenesis (Théry et al., 2002), suggesting that inhibitors of receptor tyrosine kinases may act at this step. How FGFR1 is positioned in the exosome membrane (inside or out), how FGF2 binds FGFR1 in exosomes, and how exosomal FGF2 activates FGFR1 in leukemia cells, are areas of active investigation.

FGF2 has been previously implicated in hematologic malignancy progression and development of resistance (Sato et al., 2002; Chesi et al., 2001). Elevated levels of FGF2 have previously been measured in the serum of CML and AML patients (Aguayo et al., 2000; Aguayo et al., 2002), as well as in the bone marrow of AML patients, where it was reported to function as an autocrine promotor of proliferation (Bieker et al., 2003). We found that FGF2 expression was increased in CML and AML stroma during the development of resistance to kinase inhibitors, indicating that FGF2 expression is a regulated autocrine growth factor for stroma (Traer et al., 2016). This is consistent with the role of FGF2-FGFR1 autocrine expansion of stroma in stress-induced hematopoiesis (Itkin et al., 2012; Zhao et al., 2012) and suggests that leukemia cells are able to hijack the FGF2 stress response for survival. The regulation of FGF2-FGFR1 signaling is also supported by the positive correlation in expression of both FGF2 and FGFR1 in a subset of primary AML marrow samples (Figure 4G), indicating that this pathway can be selectively activated. FGFR inhibitors not only inhibit autocrine growth of stroma, but reduce exosome secretion and significantly alter the protective ability of stromal cells (Figures 4A and 7). Since exosomes contain a complex mixture of proteins, cytokines, lipids and microRNAs (all of which potentially contribute to leukemia cell protection), inhibiting secretion of exosomes is a promising approach to blunting this complex mechanism of resistance.

In summary, FGF2 is a regulated autocrine growth factor for marrow stroma that is important in reprogramming the marrow stroma during development of resistance to TKIs. FGF2-FGFR1 activation in marrow stroma leads to increased secretion of exosomes, which are protective of leukemia cells in both in vitro and in vivo models. Given the inevitable development of clinical resistance to TKIs (FLT3 ITD AML in particular), addition of FGFR inhibitors to directly modulate the leukemia niche is a promising approach to improve the durability of response.

All data generated or analysed during this study are included in the manuscript and supporting files. Movies for confocal images in Figures 1 and 7 is provided.

1. 1. Agarwal A
   2. Eide CA
   3. Harlow A
   4. Corbin AS
   5. Mauro MJ
   6. Druker BJ
   7. Corless CL
   8. Heinrich MC
   9. Deininger MW

   (2008) An activating KRAS mutation in imatinib-resistant chronic myeloid leukemia

   Leukemia 22:2269–2272.

   https://doi.org/10.1038/leu.2008.124

   • PubMed
   • Google Scholar
2. 1. Aguayo A
   2. Kantarjian H
   3. Manshouri T
   4. Gidel C
   5. Estey E
   6. Thomas D
   7. Koller C
   8. Estrov Z
   9. O'Brien S
   10. Keating M
   11. Freireich E
   12. Albitar M

   (2000)

   Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes

   Blood 96:2240–2245.

   • PubMed
   • Google Scholar
3. 1. Aguayo A
   2. Kantarjian HM
   3. Estey EH
   4. Giles FJ
   5. Verstovsek S
   6. Manshouri T
   7. Gidel C
   8. O'Brien S
   9. Keating MJ
   10. Albitar M

   (2002) Plasma vascular endothelial growth factor levels have prognostic significance in patients with acute myeloid leukemia but not in patients with myelodysplastic syndromes

   Cancer 95:1923–1930.

   https://doi.org/10.1002/cncr.10900

   • PubMed
   • Google Scholar
4. 1. Amorim M
   2. Fernandes G
   3. Oliveira P
   4. Martins-de-Souza D
   5. Dias-Neto E
   6. Nunes D

   (2014) The overexpression of a single oncogene (ERBB2/HER2) alters the proteomic landscape of extracellular vesicles

   Proteomics 14:1472–1479.

   https://doi.org/10.1002/pmic.201300485

   • PubMed
   • Google Scholar
5. 1. Avraham H
   2. Banu N
   3. Scadden DT
   4. Abraham J
   5. Groopman JE

   (1994)

   Modulation of megakaryocytopoiesis by human basic fibroblast growth factor

   Blood 83:2126–2132.

   • PubMed
   • Google Scholar
6. 1. Ayala F
   2. Dewar R
   3. Kieran M
   4. Kalluri R

   (2009) Contribution of bone microenvironment to leukemogenesis and leukemia progression

   Leukemia 23:2233–2241.

   https://doi.org/10.1038/leu.2009.175

   • PubMed
   • Google Scholar
7. 1. Berardi AC
   2. Wang A
   3. Abraham J
   4. Scadden DT

   (1995)

   Basic fibroblast growth factor mediates its effects on committed myeloid progenitors by direct action and has no effect on hematopoietic stem cells

   Blood 86:2123–2129.

   • PubMed
   • Google Scholar
8. 1. Bianchi G
   2. Banfi A
   3. Mastrogiacomo M
   4. Notaro R
   5. Luzzatto L
   6. Cancedda R
   7. Quarto R

   (2003) Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2

   Experimental Cell Research 287:98–105.

   https://doi.org/10.1016/S0014-4827(03)00138-1

   • PubMed
   • Google Scholar
9. 1. Bieker R
   2. Padró T
   3. Kramer J
   4. Steins M
   5. Kessler T
   6. Retzlaff S
   7. Herrera F
   8. Kienast J
   9. Berdel WE
   10. Mesters RM

   (2003)

   Overexpression of basic fibroblast growth factor and autocrine stimulation in acute myeloid leukemia

   Cancer Research 63:7241–7246.

   • PubMed
   • Google Scholar
10. 1. Chesi M
    2. Brents LA
    3. Ely SA
    4. Bais C
    5. Robbiani DF
    6. Mesri EA
    7. Kuehl WM
    8. Bergsagel PL

    (2001) Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma

    Blood 97:729–736.

    https://doi.org/10.1182/blood.V97.3.729

    • PubMed
    • Google Scholar
11. 1. Choi JS
    2. Yoon HI
    3. Lee KS
    4. Choi YC
    5. Yang SH
    6. Kim IS
    7. Cho YW

    (2016) Exosomes from differentiating human skeletal muscle cells trigger myogenesis of stem cells and provide biochemical cues for skeletal muscle regeneration

    Journal of Controlled Release 222:107–115.

    https://doi.org/10.1016/j.jconrel.2015.12.018

    • PubMed
    • Google Scholar
12. 1. Colmone A
    2. Amorim M
    3. Pontier AL
    4. Wang S
    5. Jablonski E
    6. Sipkins DA

    (2008) Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells

    Science 322:1861–1865.

    https://doi.org/10.1126/science.1164390

    • PubMed
    • Google Scholar
13. 1. Coutu DL
    2. François M
    3. Galipeau J

    (2011) Inhibition of cellular senescence by developmentally regulated FGF receptors in mesenchymal stem cells

    Blood 117:6801–6812.

    https://doi.org/10.1182/blood-2010-12-321539

    • PubMed
    • Google Scholar
14. 1. Filipazzi P
    2. Bürdek M
    3. Villa A
    4. Rivoltini L
    5. Huber V

    (2012) Recent advances on the role of tumor exosomes in immunosuppression and disease progression

    Seminars in Cancer Biology 22:342–349.

    https://doi.org/10.1016/j.semcancer.2012.02.005

    • PubMed
    • Google Scholar
15. 1. Ghiaur G
    2. Levis M

    (2017) Mechanisms of resistance to FLT3 inhibitors and the role of the bone marrow microenvironment

    Hematology/Oncology Clinics of North America 31:681–692.

    https://doi.org/10.1016/j.hoc.2017.04.005

    • PubMed
    • Google Scholar
16. 1. Guagnano V
    2. Furet P
    3. Spanka C
    4. Bordas V
    5. Le Douget M
    6. Stamm C
    7. Brueggen J
    8. Jensen MR
    9. Schnell C
    10. Schmid H
    11. Wartmann M
    12. Berghausen J
    13. Drueckes P
    14. Zimmerlin A
    15. Bussiere D
    16. Murray J
    17. Graus Porta D

    (2011) Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), a potent and selective inhibitor of the fibroblast growth factor receptor family of receptor tyrosine kinase

    Journal of Medicinal Chemistry 54:7066–7083.

    https://doi.org/10.1021/jm2006222

    • PubMed
    • Google Scholar
17. 1. Huan J
    2. Hornick NI
    3. Goloviznina NA
    4. Kamimae-Lanning AN
    5. David LL
    6. Wilmarth PA
    7. Mori T
    8. Chevillet JR
    9. Narla A
    10. Roberts CT
    11. Loriaux MM
    12. Chang BH
    13. Kurre P

    (2015) Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes

    Leukemia 29:2285–2295.

    https://doi.org/10.1038/leu.2015.163

    • PubMed
    • Google Scholar
18. 1. Huang JC
    2. Basu SK
    3. Zhao X
    4. Chien S
    5. Fang M
    6. Oehler VG
    7. Appelbaum FR
    8. Becker PS

    (2015) Mesenchymal stromal cells derived from acute myeloid leukemia bone marrow exhibit aberrant cytogenetics and cytokine elaboration

    Blood Cancer Journal 5:e302.

    https://doi.org/10.1038/bcj.2015.17

    • PubMed
    • Google Scholar
19. 1. Itkin T
    2. Ludin A
    3. Gradus B
    4. Gur-Cohen S
    5. Kalinkovich A
    6. Schajnovitz A
    7. Ovadya Y
    8. Kollet O
    9. Canaani J
    10. Shezen E
    11. Coffin DJ
    12. Enikolopov GN
    13. Berg T
    14. Piacibello W
    15. Hornstein E
    16. Lapidot T

    (2012) FGF-2 expands murine hematopoietic stem and progenitor cells via proliferation of stromal cells, c-Kit activation, and CXCL12 down-regulation

    Blood 120:1843–1855.

    https://doi.org/10.1182/blood-2011-11-394692

    • PubMed
    • Google Scholar
20. 1. Javidi-Sharifi N
    2. Traer E
    3. Martinez J
    4. Gupta A
    5. Taguchi T
    6. Dunlap J
    7. Heinrich MC
    8. Corless CL
    9. Rubin BP
    10. Druker BJ
    11. Tyner JW

    (2015) Crosstalk between KIT and FGFR3 promotes gastrointestinal stromal tumor cell growth and drug resistance

    Cancer Research 75:880–891.

    https://doi.org/10.1158/0008-5472.CAN-14-0573

    • PubMed
    • Google Scholar
21. 1. Manshouri T
    2. Estrov Z
    3. Quintás-Cardama A
    4. Burger J
    5. Zhang Y
    6. Livun A
    7. Knez L
    8. Harris D
    9. Creighton CJ
    10. Kantarjian HM
    11. Verstovsek S

    (2011) Bone marrow stroma-secreted cytokines protect JAK2(V617F)-mutated cells from the effects of a JAK2 inhibitor

    Cancer Research 71:3831–3840.

    https://doi.org/10.1158/0008-5472.CAN-10-4002

    • PubMed
    • Google Scholar
22. 1. Marlein CR
    2. Zaitseva L
    3. Piddock RE
    4. Robinson SD
    5. Edwards DR
    6. Shafat MS
    7. Zhou Z
    8. Lawes M
    9. Bowles KM
    10. Rushworth SA

    (2017) NADPH oxidase-2 derived superoxide drives mitochondrial transfer from bone marrow stromal cells to leukemic blasts

    Blood 130:1649–1660.

    https://doi.org/10.1182/blood-2017-03-772939

    • PubMed
    • Google Scholar
23. 1. Meads MB
    2. Gatenby RA
    3. Dalton WS

    (2009) Environment-mediated drug resistance: a major contributor to minimal residual disease

    Nature Reviews Cancer 9:665–674.

    https://doi.org/10.1038/nrc2714

    • PubMed
    • Google Scholar
24. 1. Mohammadi M
    2. Froum S
    3. Hamby JM
    4. Schroeder MC
    5. Panek RL
    6. Lu GH
    7. Eliseenkova AV
    8. Green D
    9. Schlessinger J
    10. Hubbard SR

    (1998) Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain

    The EMBO Journal 17:5896–5904.

    https://doi.org/10.1093/emboj/17.20.5896

    • PubMed
    • Google Scholar
25. 1. Moschoi R
    2. Imbert V
    3. Nebout M
    4. Chiche J
    5. Mary D
    6. Prebet T
    7. Saland E
    8. Castellano R
    9. Pouyet L
    10. Collette Y
    11. Vey N
    12. Chabannon C
    13. Recher C
    14. Sarry JE
    15. Alcor D
    16. Peyron JF
    17. Griessinger E

    (2016) Protective mitochondrial transfer from bone marrow stromal cells to acute myeloid leukemic cells during chemotherapy

    Blood 128:253–264.

    https://doi.org/10.1182/blood-2015-07-655860

    • PubMed
    • Google Scholar
26. 1. Paggetti J
    2. Haderk F
    3. Seiffert M
    4. Janji B
    5. Distler U
    6. Ammerlaan W
    7. Kim YJ
    8. Adam J
    9. Lichter P
    10. Solary E
    11. Berchem G
    12. Moussay E

    (2015) Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

    Blood 126:1106–1117.

    https://doi.org/10.1182/blood-2014-12-618025

    • PubMed
    • Google Scholar
27. 1. Parmar A
    2. Marz S
    3. Rushton S
    4. Holzwarth C
    5. Lind K
    6. Kayser S
    7. Döhner K
    8. Peschel C
    9. Oostendorp RA
    10. Götze KS

    (2011) Stromal niche cells protect early leukemic FLT3-ITD+ progenitor cells against first-generation FLT3 tyrosine kinase inhibitors

    Cancer Research 71:4696–4706.

    https://doi.org/10.1158/0008-5472.CAN-10-4136

    • PubMed
    • Google Scholar
28. 1. Peinado H
    2. Alečković M
    3. Lavotshkin S
    4. Matei I
    5. Costa-Silva B
    6. Moreno-Bueno G
    7. Hergueta-Redondo M
    8. Williams C
    9. García-Santos G
    10. Ghajar C
    11. Nitadori-Hoshino A
    12. Hoffman C
    13. Badal K
    14. Garcia BA
    15. Callahan MK
    16. Yuan J
    17. Martins VR
    18. Skog J
    19. Kaplan RN
    20. Brady MS
    21. Wolchok JD
    22. Chapman PB
    23. Kang Y
    24. Bromberg J
    25. Lyden D

    (2012) Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

    Nature Medicine 18:883–891.

    https://doi.org/10.1038/nm.2753

    • PubMed
    • Google Scholar
29. 1. Perez-Torres M
    2. Valle BL
    3. Maihle NJ
    4. Negron-Vega L
    5. Nieves-Alicea R
    6. Cora EM

    (2008) Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells

    Experimental Cell Research 314:2907–2918.

    https://doi.org/10.1016/j.yexcr.2008.07.013

    • PubMed
    • Google Scholar
30. 1. Proia P
    2. Schiera G
    3. Mineo M
    4. Ingrassia AM
    5. Santoro G
    6. Savettieri G
    7. Di Liegro I

    (2008) Astrocytes shed extracellular vesicles that contain fibroblast growth factor-2 and vascular endothelial growth factor

    International Journal of Molecular Medicine 21:63–67.

    https://doi.org/10.3892/ijmm.21.1.63

    • PubMed
    • Google Scholar
31. 1. Roecklein BA
    2. Torok-Storb B

    (1995)

    Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes

    Blood 85:997–1005.

    • PubMed
    • Google Scholar
32. 1. Sanderson MP
    2. Keller S
    3. Alonso A
    4. Riedle S
    5. Dempsey PJ
    6. Altevogt P

    (2008) Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion

    Journal of Cellular Biochemistry 103:1783–1797.

    https://doi.org/10.1002/jcb.21569

    • PubMed
    • Google Scholar
33. 1. Sanjana NE
    2. Shalem O
    3. Zhang F

    (2014) Improved vectors and genome-wide libraries for CRISPR screening

    Nature Methods 11:783–784.

    https://doi.org/10.1038/nmeth.3047

    • PubMed
    • Google Scholar
34. 1. Sato N
    2. Hattori Y
    3. Wenlin D
    4. Yamada T
    5. Kamata T
    6. Kakimoto T
    7. Okamoto S
    8. Kawamura C
    9. Kizaki M
    10. Shimada N
    11. Ote Y
    12. Hata J
    13. Ikeda Y

    (2002) Elevated level of plasma basic fibroblast growth factor in multiple myeloma correlates with increased disease activity

    Japanese Journal of Cancer Research 93:459–466.

    https://doi.org/10.1111/j.1349-7006.2002.tb01278.x

    • PubMed
    • Google Scholar
35. 1. Schäfer T
    2. Zentgraf H
    3. Zehe C
    4. Brügger B
    5. Bernhagen J
    6. Nickel W

    (2004) Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells

    Journal of Biological Chemistry 279:6244–6251.

    https://doi.org/10.1074/jbc.M310500200

    • PubMed
    • Google Scholar
36. 1. Schindelin J
    2. Arganda-Carreras I
    3. Frise E
    4. Kaynig V
    5. Longair M
    6. Pietzsch T
    7. Preibisch S
    8. Rueden C
    9. Saalfeld S
    10. Schmid B
    11. Tinevez JY
    12. White DJ
    13. Hartenstein V
    14. Eliceiri K
    15. Tomancak P
    16. Cardona A

    (2012) Fiji: an open-source platform for biological-image analysis

    Nature Methods 9:676–682.

    https://doi.org/10.1038/nmeth.2019

    • PubMed
    • Google Scholar
37. 1. Smith CC
    2. Wang Q
    3. Chin CS
    4. Salerno S
    5. Damon LE
    6. Levis MJ
    7. Perl AE
    8. Travers KJ
    9. Wang S
    10. Hunt JP
    11. Zarrinkar PP
    12. Schadt EE
    13. Kasarskis A
    14. Kuriyan J
    15. Shah NP

    (2012) Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia

    Nature 485:260–263.

    https://doi.org/10.1038/nature11016

    • PubMed
    • Google Scholar
38. 1. Smith CC
    2. Paguirigan A
    3. Jeschke GR
    4. Lin KC
    5. Massi E
    6. Tarver T
    7. Chin CS
    8. Asthana S
    9. Olshen A
    10. Travers KJ
    11. Wang S
    12. Levis MJ
    13. Perl AE
    14. Radich JP
    15. Shah NP

    (2017) Heterogeneous resistance to quizartinib in acute myeloid leukemia revealed by single-cell analysis

    Blood 130:48–58.

    https://doi.org/10.1182/blood-2016-04-711820

    • PubMed
    • Google Scholar
39. 1. Steringer JP
    2. Müller HM
    3. Nickel W

    (2015) Unconventional secretion of fibroblast growth factor 2--a novel type of protein translocation across membranes?

    Journal of Molecular Biology 427:1202–1210.

    https://doi.org/10.1016/j.jmb.2014.07.012

    • PubMed
    • Google Scholar
40. 1. Théry C
    2. Zitvogel L
    3. Amigorena S

    (2002) Exosomes: composition, biogenesis and function

    Nature Reviews Immunology 2:569–579.

    https://doi.org/10.1038/nri855

    • PubMed
    • Google Scholar
41. 1. Traer E
    2. MacKenzie R
    3. Snead J
    4. Agarwal A
    5. Eiring AM
    6. O'Hare T
    7. Druker BJ
    8. Deininger MW

    (2012) Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors

    Leukemia 26:1140–1143.

    https://doi.org/10.1038/leu.2011.325

    • PubMed
    • Google Scholar
42. 1. Traer E
    2. Javidi-Sharifi N
    3. Agarwal A
    4. Dunlap J
    5. English I
    6. Martinez J
    7. Tyner JW
    8. Wong M
    9. Druker BJ

    (2014) Ponatinib overcomes FGF2-mediated resistance in CML patients without kinase domain mutations

    Blood 123:1516–1524.

    https://doi.org/10.1182/blood-2013-07-518381

    • PubMed
    • Google Scholar
43. 1. Traer E
    2. Martinez J
    3. Javidi-Sharifi N
    4. Agarwal A
    5. Dunlap J
    6. English I
    7. Kovacsovics T
    8. Tyner JW
    9. Wong M
    10. Druker BJ

    (2016) FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

    Cancer Research 76:6471–6482.

    https://doi.org/10.1158/0008-5472.CAN-15-3569

    • PubMed
    • Google Scholar
44. 1. Trudel S
    2. Ely S
    3. Farooqi Y
    4. Affer M
    5. Robbiani DF
    6. Chesi M
    7. Bergsagel PL

    (2004) Inhibition of fibroblast growth factor receptor 3 induces differentiation and apoptosis in t(4;14) myeloma

    Blood 103:3521–3528.

    https://doi.org/10.1182/blood-2003-10-3650

    • PubMed
    • Google Scholar
45. 1. Viola S
    2. Traer E
    3. Huan J
    4. Hornick NI
    5. Tyner JW
    6. Agarwal A
    7. Loriaux M
    8. Johnstone B
    9. Kurre P

    (2016) Alterations in acute myeloid leukaemia bone marrow stromal cell exosome content coincide with gains in tyrosine kinase inhibitor resistance

    British Journal of Haematology 172:983–986.

    https://doi.org/10.1111/bjh.13551

    • PubMed
    • Google Scholar
46. 1. Ware KE
    2. Hinz TK
    3. Kleczko E
    4. Singleton KR
    5. Marek LA
    6. Helfrich BA
    7. Cummings CT
    8. Graham DK
    9. Astling D
    10. Tan AC
    11. Heasley LE

    (2013) A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop

    Oncogenesis 2:e39.

    https://doi.org/10.1038/oncsis.2013.4

    • PubMed
    • Google Scholar
47. 1. Weisberg E
    2. Wright RD
    3. McMillin DW
    4. Mitsiades C
    5. Ray A
    6. Barrett R
    7. Adamia S
    8. Stone R
    9. Galinsky I
    10. Kung AL
    11. Griffin JD

    (2008) Stromal-mediated protection of tyrosine kinase inhibitor-treated BCR-ABL-expressing leukemia cells

    Molecular Cancer Therapeutics 7:1121–1129.

    https://doi.org/10.1158/1535-7163.MCT-07-2331

    • PubMed
    • Google Scholar
48. 1. Weisberg E
    2. Barrett R
    3. Liu Q
    4. Stone R
    5. Gray N
    6. Griffin JD

    (2009) FLT3 inhibition and mechanisms of drug resistance in mutant FLT3-positive AML

    Drug Resistance Updates 12:81–89.

    https://doi.org/10.1016/j.drup.2009.04.001

    • PubMed
    • Google Scholar
49. 1. Wilson TR
    2. Fridlyand J
    3. Yan Y
    4. Penuel E
    5. Burton L
    6. Chan E
    7. Peng J
    8. Lin E
    9. Wang Y
    10. Sosman J
    11. Ribas A
    12. Li J
    13. Moffat J
    14. Sutherlin DP
    15. Koeppen H
    16. Merchant M
    17. Neve R
    18. Settleman J

    (2012) Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

    Nature 487:505–509.

    https://doi.org/10.1038/nature11249

    • PubMed
    • Google Scholar
50. 1. Yang X
    2. Sexauer A
    3. Levis M

    (2014) Bone marrow stroma-mediated resistance to FLT3 inhibitors in FLT3-ITD AML is mediated by persistent activation of extracellular regulated kinase

    British Journal of Haematology 164:61–72.

    https://doi.org/10.1111/bjh.12599

    • PubMed
    • Google Scholar
51. 1. Zacherl S
    2. La Venuta G
    3. Müller HM
    4. Wegehingel S
    5. Dimou E
    6. Sehr P
    7. Lewis JD
    8. Erfle H
    9. Pepperkok R
    10. Nickel W

    (2015) A direct role for ATP1A1 in unconventional secretion of fibroblast growth factor 2

    Journal of Biological Chemistry 290:3654–3665.

    https://doi.org/10.1074/jbc.M114.590067

    • PubMed
    • Google Scholar
52. 1. Zarrinkar PP
    2. Gunawardane RN
    3. Cramer MD
    4. Gardner MF
    5. Brigham D
    6. Belli B
    7. Karaman MW
    8. Pratz KW
    9. Pallares G
    10. Chao Q
    11. Sprankle KG
    12. Patel HK
    13. Levis M
    14. Armstrong RC
    15. James J
    16. Bhagwat SS

    (2009) AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML)

    Blood 114:2984–2992.

    https://doi.org/10.1182/blood-2009-05-222034

    • PubMed
    • Google Scholar
53. 1. Zhao M
    2. Ross JT
    3. Itkin T
    4. Perry JM
    5. Venkatraman A
    6. Haug JS
    7. Hembree MJ
    8. Deng CX
    9. Lapidot T
    10. He XC
    11. Li L

    (2012) FGF signaling facilitates postinjury recovery of mouse hematopoietic system

    Blood 120:1831–1842.

    https://doi.org/10.1182/blood-2011-11-393991

    • PubMed
    • Google Scholar
54. 1. Zhou M
    2. Sutliff RL
    3. Paul RJ
    4. Lorenz JN
    5. Hoying JB
    6. Haudenschild CC
    7. Yin M
    8. Coffin JD
    9. Kong L
    10. Kranias EG
    11. Luo W
    12. Boivin GP
    13. Duffy JJ
    14. Pawlowski SA
    15. Doetschman T

    (1998) Fibroblast growth factor 2 control of vascular tone

    Nature Medicine 4:201–207.

    https://doi.org/10.1038/nm0298-201

    • PubMed
    • Google Scholar

[Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed.]

Thank you for submitting your article "FGF2-FGFR1 signaling regulates release of leukemia-protective exosomes from bone marrow stromal cells" for consideration by eLife. Your article has been reviewed by two peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jeffrey Settleman as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Thomas O'Hare (Reviewer #2).

The Reviewing Editor has highlighted the concerns that require revision and/or responses, and we have included the separate reviews below for your consideration. If you have any questions, please do not hesitate to contact us.

Summary:

This manuscript addresses the mechanism(s) by which protective signaling from the leukemia microenvironment may promote leukemia cell persistence, the development of drug resistance, and ultimately disease relapse. The authors claim that fibroblast growth factor 2 (FGF2) is released in exosomes from bone marrow stromal cells and that the subsequent endocytic entry of FGF2 into leukemia cells protects the leukemia cells from oncoprotein kinase targeted inhibitors. The authors claim that expression of FGF2, and its receptor FGFR1, is increased in a subset of stromal cell lines and primary AML stroma. Moreover, they claim that increased FGF2/FGFR1 signaling is associated with increased exosome secretion such that pharmacologic or genetic inhibition of FGFR1 signaling inhibition interrupts stromal autocrine growth and decreases secretion of FGF2-containing exosomes resulting in decreased protection of leukemia cells. Support for this hypothesis comes from the observation that, when BCR-ABL-driven leukemia cells are transplanted into FGF2-deficient mice, they display enhanced therapeutic benefit compared to FGF2-proficient mice. Hence, the authors claim that inhibition of the FGF2->FGFR1 signaling axis may be a strategy to prevent or overcome the development of TKI resistance in leukemia. Overall, the findings of this study are supported by strong, well-controlled experimental data and the manuscript is clearly written. However, both reviewers have both major and minor concerns, which if addressed by the authors, will substantially improve the quality of the manuscript.

Separate reviews (please respond to each point):

Reviewer #1:

This manuscript address the mechanism(s) by which protective signaling from the leukemia microenvironment may promote leukemia cell persistence, the development of drug resistance, and ultimately disease relapse. The authors claim that fibroblast growth factor 2 (FGF2) is released in exosomes from bone marrow stromal cells and that the subsequent endocytic entry of FGF2 into leukemia cells protects the leukemia cells from oncoprotein kinase targeted inhibitors. The authors claim that expression of FGF2 and its receptor FGFR1, are increased in a subset of stromal cell lines and primary AML stroma. Moreover, they claim that increased FGF2/FGFR1 signaling is associated with increased exosome secretion such that pharmacologic or genetic inhibition of FGFR1 signaling inhibition interrupts stromal autocrine growth and decreases secretion of FGF2-containing exosomes resulting in decreased protection of leukemia cells. Support for this hypothesis comes from the observation that, when BCR-ABL driven leukemia cells are transplanted into FGF2 deficient mice, they display enhanced therapeutic benefit compared to FGF2 proficient mice. Hence, the authors claim that inhibition of the FGF2->FGFR1 signaling axis may be a strategy to prevent or overcome the development of TKI resistance in leukemia.

The authors conclude from the data in Figure 1C that ECVs produced by HS-5 cells are internalized by endocytosis. What evidence do the authors have the ECVs are actually entering cells by endocytosis and, if entry is by endocytosis, is it clathrin-mediated endocytosis, caveolae, micropinocytosis or phagocytosis?

The data in Figures2E/F on protection of exosomal FGF2 from proteinase K digestion are quite equivocal in that the partial protection observed is not entirely consistent with the authors' interpretations. Indeed, the protection described in Figure 2F is so marginal as to make this reviewer wonder how reproducible the data are and whether, or not, a modest increase in incubation time with proteinase K would lead to complete proteolysis of FGF2. Indeed, the authors' conclusions that, "….. FGF2 is contained within exosomes but also likely associated with proteins on the surface of exosomes." is rather ambivalent.

In Figure 3, the authors convincingly demonstrate that HS-5 CM contains more exosomes that HS-27 CM. They also note that the receptor for FGF2, FGFR1, is also present in exosomes. One obvious question is whether FGFR1 is tyrosine phosphorylated on sites that are stimulated by FGF2, which would suggest that a portion of FGF2 and FGFR1 are in complex with one another.

In Figure 5, the authors show that TKIs of FGFR1 signaling reduce exosome release from HS-5 stromal cells. This data are convincing, but it is also rather phenomenological and lacks a mechanism by which FGFR1 might regulate the release of exosomes by bone marrow stromal cells. Also, the fact that this occurs in one cell line does not lend confidence that this is a generalizable conclusion.

In Figure 6, the use of Tet-regulated shRNA targeting shRNA is a good complement to the use of pharmacological TKIs of FGFR1 signaling. However, the effects of shFGFR1 on exosomal FGF2 should be assessed in Figure 6B – since the effects of shFGFR1 on tsg101 and CD9 are barely discernible given the poor quality of immunoblots presented in this figure. Furthermore, the authors need to better explain why their CRISPR/CAS9 efforts to silence the expression of either FGFR1 or FGF2 resulted in partial loss of FGFR1 or FGF2 expression. Moreover, whereas partial silencing of FGFR1 had almost no discernible effect on exosomal tsg101 or CD9, partial silencing of FGF2 had an appreciable effect on exosomal tsg101 and CD9. These data are somewhat discordant and are not adequately explained in the text.

The experiments presented in Figure 7 were nicely conceived and elegantly executed. This figure makes a case for an important role of stromal derived, exosome delivered FGF2 as a relevant mechanism of TKI resistance in leukemia.

Reviewer #2:

The findings of the study are supported by strong, well-controlled experimental data. The paper is clearly written. My major concerns are the heavy reliance on HS-5 as proxy for the leukemic microenvironment and that the story is almost entirely presented from the perspective of the stroma. Several mechanistic questions pertaining to the leukemic cells are asked below, with the goal of helping the reader to better understand how the authors are viewing and rationalizing these points.

1) A stated theme of the study is that the normal hematopoietic microenvironment is altered by leukemia. While this is undoubtedly correct, the choice of CM from a cell line derived from normal bone marrow would seem to limit applicability and relevance to the leukemic microenvironment.

2) Why doesn't the presence of an FGFR inhibitor (PD173074) in experiments in which CM is provided to leukemic cell lines dampen the effectiveness of HS-5 CM? The authors make a convincing case that FGF2-laden exosomes are taken up by the leukemic cell lines and that this provides a protective effect against TKIs. They also show that FGFR inhibition (or [partial] FGFR1 deletion) in HS-5 stromal cells decreases exosome production/secretion and probably alters the content of exosomes. However, the story from the point of view of the leukemic cell lines is largely unexplored and mechanistically vague. Does FGFR1 deletion in the leukemic cell lines change the effectiveness of HS-5 CM? Does PD173074 completely shut off FGFR1 signaling in the leukemic cell lines? Perhaps I am missing something, but I find it surprising that inhibition of FGFR1 (in the leukemic cells) does not at least partially reverse the protective effects of HS-5 CM. This is largely based on the authors' reasonable assertion that the most important exosome cargo is FGF2.

3) Several TKIs used in CML have target profiles that include FGFR, most notably the pan-FGFR TKI, ponatinib. Midostaurin, the currently approved frontline TKI for FLT3-mutated AML, inhibits FGFR1 and 2. Would patients treated with these TKIs derive additional benefit from collateral inhibition of FGFR1 in stromal cells (and possibly in leukemic cells)? I expected to see a comparison of imatinib and ponatinib in the study. I also thought this would be worth testing in the mouse model, as mentioned below.

4) Panels 4F,G show data for primary AML stromal cells (n = 42). The legend should specify how many AML patients are represented and how many data points are from each patient. Are some of these FLT3-ITD patients, and if so, can you indicate them as colored dots? This display item and the corresponding Materials and methods, Results and Discussion require more detail. This has potential to be an intriguing part of the story but the presentation is murky. Is it possible to include a comparison to HS-5 and HS-27 in these panels? Are the FGFR1 and FGF2 levels (and their correlations) substantially different from normal bone marrow? Should we expect them to be? Please consider strengthening this part of the manuscript. I could not glean much as presented.

Minor Comments:

1) Figure 1: In panel A, what is the result if the same experiment is carried out with ponatinib (10-25 nM)? dasatinib (10-25 nM)? In panel B, with midostaurin (1 µM)? This comment is somewhat redundant with major point (3) above; here, I am requesting that the data are included, at least in the supplement.

2) Why was 1 µM imatinib used? This concentration, at least at the 48 h timepoint, does not reduce proliferation (Figure 1A) to the extent that AC220 does in MOLM14 cells. Thus, it is difficult to determine the extent to which CM provides a protective effect. What happens if a higher concentration of imatinib is used, or if a more potent TKI is used at a concentration that matches the AC220 magnitude of reduced proliferation?

3) Regarding the FGFR inhibitors:

– Why two? BGJ-398 is described as 'selective' but I did not find the rationale for needing it in the study (which began with PD173074).

– What is the direct evidence/readout to confirm that these inhibitors are working (and to measure the extent of inhibition)?

4) Perhaps it's not a reasonable comparison, but it is surprising that shRNA-based elimination of FGFR1 does not reduce FGF2 levels in HS-5 cells (Figure 6A), but FGFR inhibition does (Figure 5C). Also, the HS-5 CRISPR FGFR1.1 and 1.2 lines show reduced FGF2. The HS-5 CRISPR FGF2.1 and 2.2 lines show enhanced FGFR1 but reduced FGFR1 in ECVs. Can the authors comment on / explain these points?

5) In Figure 6B, I would like to be able to evaluate whether reduced FGFR1 brings about a reduction in FGF2, but this data is not provided. In general, both FGF2 and FGFR1 should be shown in immunoblot stacks. Sometimes, it is one or the other.

6) Figure 7:

– why is nilotinib used in 7A? All other data are with imatinib.

– the main point of the paper seems to be that interrupting the FGFR1-FGF2 network in stromal cells would decrease the protective effects emanating from the microenvironment. To test this, shouldn't arms with "PD PD173074" alone, and with "nilotinib + PD173074" be included?

– why did you choose this model rather than a FLT3-ITD model? The jumping back and forth between CML and AML is distracting. Could the authors consider a focus on MOLM14 and FLT3-ITD driven AML in the main manuscript, with the CML work allocated to the supplement?

7) Please address the following clerical issues:

– abbreviations are defined several times (examples: TKI; conditioned media)

– conventions are not consistently followed (ml vs. mL; h vs. hrs vs. hours; spacing between value and unit [e.g. 10nM vs. 10 nM]; no spacing vs. spacing preceding a citation)

– Chemical formulas lack proper subscripting (possibly an inherent submission portal limitation)

– Please check supplier names (Genscript or GenScript? Is Jackson laboratories the correct name of this supplier?)

– References 14 and 30 are identical

– A few article titles in References section are title case (#4, 15, 16, for example)

– Reference 23 lacks page number, etc. The final manuscript is 2016, not 2015

– Reference 25 lacks journal name, etc.

– Figure 2 legend: NB40 or NP-40?

– Final paragraph of subsection “FGF2 is Contained in Stromal Cell ECVs and Exosomes”: "membrate"

– First paragraph of subsection “FGF2-Fgfr1 Signaling Promotes Stromal Growth and Paracrine Protection of Leukemia”: "withg"

– "proteinase K" vs. "Proteinase K"

– "PD173074" vs. "PD-173074"

– Second paragraph of the Discussion: a callout to Figure 3E is made. Should this be 3D (there is no panel 3E)?

Additional data files and statistical comments:

I asked the authors to consider including additional data based on experiments with tyrosine kinase inhibitors beyond imatinib and AC220. If they choose to do this, inclusion of the data files is warranted. I do not see the need for any other additional data files.

Statistical analysis and description of statistical methods is adequate and appropriate.

Experimental rigor is evident in the work presented in the manuscript.

Reviewer #1:

[…] The authors conclude from the data in Figure 1C that ECVs produced by HS-5 cells are internalized by endocytosis. What evidence do the authors have the ECVs are actually entering cells by endocytosis and, if entry is by endocytosis, is it clathrin-mediated endocytosis, caveolae, micropinocytosis or phagocytosis?

The reviewer makes a good point, we do not present evidence that internalization is mediated by endocytosis. More detailed mechanistic studies of exosome transfer and uptake is in progress and beyond the scope of this manuscript. The text was modified as follows:

Analysis by confocal microscopy showed that ECVs are indeed internalized by leukemia cells, although the exact mechanism of internalization is still under investigation (Figure 1C and Supplemental movies).

The data in Figures2E/F on protection of exosomal FGF2 from proteinase K digestion are quite equivocal in that the partial protection observed is not entirely consistent with the authors' interpretations. Indeed, the protection described in Figure 2F is so marginal as to make this reviewer wonder how reproducible the data are and whether, or not, a modest increase in incubation time with proteinase K would lead to complete proteolysis of FGF2. Indeed, the authors' conclusions that, "….. FGF2 is contained within exosomes but also likely associated with proteins on the surface of exosomes." is rather ambivalent.

We repeated this experiment multiple times and it is technically challenging. Even the intact HS-5 cells in Figure 2E (mostly intracellular FGF2) have some degraded FGF2 after proteinase K treatment, likely from cell rupture. Isolated EVs and exosomes are even more fragile and we found that even integral membrane proteins tsg-101 and CD9 were partially degraded by proteinase K. The fact that we could detect intact FGF2 after proteinase K is strong evidence that some FGF2 (and perhaps most) is contained within EVs and exosomes. The text was modified to state this more explicitly.

We conclude that FGF2 is contained within EVs and exosomes, however we cannot exclude that FGF2 may also be present on the surface since partial FGF2 degradation was noted in intact HS-5 cells, EVs and purified exosomes (Figures 2E-F).

In Figure 3, the authors convincingly demonstrate that HS-5 CM contains more exosomes that HS-27 CM. They also note that the receptor for FGF2, FGFR1, is also present in exosomes. One obvious question is whether FGFR1 is tyrosine phosphorylated on sites that are stimulated by FGF2, which would suggest that a portion of FGF2 and FGFR1 are in complex with one another.

This is something we are quite interested in, however there are no reliable phospho-FGFR1 antibodies that we have found – and not for lack of trying! To address the issue of FGF2 bound to FGFR1 directly, we are generating FGF2 and FGFR1 expression constructs with bi-fluorescent complementation tags. This system produces fluorescence when FGF2 and FGFR1 are bound to each other, bringing the bi-fluorescent tags together to generate fluorescent signal. However, we do not have this data yet.

In Figure 5, the authors show that TKIs of FGFR1 signaling reduce exosome release from HS-5 stromal cells. This data are convincing, but it is also rather phenomenological and lacks a mechanism by which FGFR1 might regulate the release of exosomes by bone marrow stromal cells. Also, the fact that this occurs in one cell line does not lend confidence that this is a generalizable conclusion.

There are limited human marrow stromal cell lines available, but HS-5 and HS-27 are widely used as representative of distinct phenotypic subsets of marrow stroma and have clear differences in FGF2/FGFR1 expression. We were able to replicate reduction in exosomes with FGFR inhibition in primary stroma from FGF2 +/+ mice but not FGF2 -/- mice (Figures 6 and 7), which we feel is the best genetic control. We attempted to treat primary human stroma with FGFR inhibitors but these cultures are quite heterogeneous making comparisons difficult, and we were unable to obtain sufficient conditioned media from primary cultures for reliable exosome quantification after FGFR inhibition.

This FGFR1 downstream pathways that regulate exosome secretion is still under investigation in our lab. Some of our preliminary data is in Author response image 1 and suggests that FGFR1 regulates exosome biogenesis through phospho-lipase C and protein kinase C activation, but experiments are ongoing to prove this.

Author response image 1

Download asset Open asset

Ongoing model and mechanistic work on PLC. We reasoned that FGFR1 can either lead to increased exosome biosynthesis and/or release. One of the well-established FGFR signaling intermediaries is phospholipase C, a membrane-associated enzyme that cleaves phospholipids into diacyl glycerol (DAG) and inositol 1,4,5-triphosphoate (IP3). Secretory vesicle trafficking involves several steps that are controlled by DAG, including the fission of vesicles at the trans-Golgi network, the generation and maturation of multivesicular bodies, and the docking and fusion at the plasma membrane. Previous reports have shown that DAG kinase α (DGKα converts DAG to phosphatidic acid) expression decreases the production of FasL-containing exosomes by T lymphocytes, and that inhibition of DGKα enhances the production (Alonso et al. Cell Death Differ. 2011). DAG also activates protein kinase C (PKC), an enzyme which is recruited to the plasma membrane or to a number of intracellular compartments upon activation. PKC controls the endocytosis, trafficking and recycling of several receptor tyrosine kinases. This may be a relevant mechanism for the regulation of endocytosis and subsequent routing to multivesicular bodies. Although PKC has not previously been shown to phosphorylate FGFR1, FGFR1 does possess C-terminal serine/threonine phosphorylation sites that are known to be important for regulation of endocytosis(Nadratowska-Wesolowska et al. Oncogene. 2014).

We therefore sought to test the roles of PLC and PKC in exosome release in HS-5 cells. A rise in intracellular calcium is necessary for regulated exocytosis of secretory granules and exosomes. We therefore used the cell-permeable calcium ionophore ionomycin as a positive control for the regulation of exosome release. PD173074, which we confirmed as a negative regulator of exosome production in previous figures, served as a negative control. Phorbol 12-myristate 13-acetate (PMA) is a mimic of DAG and activates PKC. Over a timecourse of 72 hours, both PMA and ionomycin led to increased exosome production/release. PD173074 attenuated exosome production. Interestingly, this was not rescued by combined treatment with ionomycin, suggesting that FGFR inhibits early exosome biogenesis rather than release.

In Figure 6, the use of Tet-regulated shRNA targeting shRNA is a good complement to the use of pharmacological TKIs of FGFR1 signaling. However, the effects of shFGFR1 on exosomal FGF2 should be assessed in Figure 6B – since the effects of shFGFR1 on tsg101 and CD9 are barely discernible given the poor quality of immunoblots presented in this figure. Furthermore, the authors need to better explain why their CRISPR/CAS9 efforts to silence the expression of either FGFR1 or FGF2 resulted in partial loss of FGFR1 or FGF2 expression. Moreover, whereas partial silencing of FGFR1 had almost no discernible effect on exosomal tsg101 or CD9, partial silencing of FGF2 had an appreciable effect on exosomal tsg101 and CD9. These data are somewhat discordant and are not adequately explained in the text.

The FGF2 protein in ECVs after shRNA silencing of FGFR1 was assessed by immunoblot in this experiment, but unfortunately was uninterpretable. The amount of protein in ECVs is very low, and many of our immunoblots were at the limit of detection when performing small scale experiments (i.e. not using multiple 15 cm dishes of HS-5 cells to generate ECVs). The immunoblots for tsg-101 and CD9 in Figure 6 were darkened to allow better visualization of the bands.

The CRISPR/CAS9 experiments were complicated by the fact that complete silencing of either FGFR1 or FGF2 in HS-5 cells essentially halted cell replication and we were never able to continue to culture cells with stable FGF2 and FGFR genetic deletion. We attempted this experiment numerous times, but could only analyze the cells for a short period after initial silencing. For this reason, the CRISPR/CAS9 cells only have partial silencing of FGF2 and FGFR1 protein. We did notice that FGF2 silencing led to an increase in FGFR1 protein on the cells and more rapid decrease in ECVs. The increase in FGFR1 proteins is something we have observed with FGFR inhibitors previously (Traer et al. Blood 2014 and Traer et al. Cancer Research 2016) and based upon our previous data we suspect that removing FGF2 ligand leads to less activation and internalization of FGFR1. We suspect that silencing of FGFR1 takes longer to affect ECV production since there is still abundant FGF2 ligand present to drive activity of the remaining FGFR1. Given the limitations of our genetic silencing tools, we could not investigate this more rigorously. Given the limitations of using CRISPR/CAS9 for FGF2 and FGFR1 in HS-5 cells, this data was moved to the supplement, along with a more detailed explanation of the above issues.

In contrast to the CRISPR/CAS9 data, the primary stroma from FGF2 +/+ and -/- mice is a cleaner and more robust genetic control. Although the FGF2 -/- marrow stroma does not culture well in vitro, we could overcome this deficiency by simply using more mice. The immunoblots of ECVs from primary FGF2 +/+ and -/- stroma was moved to Figure 6 from the supplement.

We feel the presentation of the data this way is easier to understand, and the issues with the CRISPR/CAS9 system – although supportive overall of the model – does have technical limitations and is more appropriate for the Supplement.

The experiments presented in Figure 7 were nicely conceived and elegantly executed. This figure makes a case for an important role of stromal derived, exosome delivered FGF2 as a relevant mechanism of TKI resistance in leukemia.

We appreciate the positive feedback, thank you.

Reviewer #2:

The findings of the study are supported by strong, well-controlled experimental data. The paper is clearly written. My major concerns are the heavy reliance on HS-5 as proxy for the leukemic microenvironment and that the story is almost entirely presented from the perspective of the stroma. Several mechanistic questions pertaining to the leukemic cells are asked below, with the goal of helping the reader to better understand how the authors are viewing and rationalizing these points.

1) A stated theme of the study is that the normal hematopoietic microenvironment is altered by leukemia. While this is undoubtedly correct, the choice of CM from a cell line derived from normal bone marrow would seem to limit applicability and relevance to the leukemic microenvironment.

Although HS-5 is derived from normal stroma, it has long been recognized has having distinct effects on hematopoiesis compared to HS-27. We previously evaluated FGF2 in stromal cells from patients with resistant CML and AML and found significantly increased FGF2 expression by immunohistochemistry during development of resistance (Traer et al. Blood 2014, Traer et al. Cancer Research 2016). The dramatically different expression of FGF2 and FGFR1 in HS-5 compared to HS-27 make it a good model to study FGF2 secretion. Also, we were unable to generate sufficient numbers of ECVs from cultured primary stromal cells from leukemia patients to make this a feasible option using currently available methods for ECV quantification.

The data with primary murine stromal cells from FGF2 +/+ and -/- and the leukemia model in Figures 6 and 7 provides strong supportive data that marrow stromal FGF2 mediates resistance in the leukemia microenvironment specifically.

2) Why doesn't the presence of an FGFR inhibitor (PD173074) in experiments in which CM is provided to leukemic cell lines dampen the effectiveness of HS-5 CM? The authors make a convincing case that FGF2-laden exosomes are taken up by the leukemic cell lines and that this provides a protective effect against TKIs. They also show that FGFR inhibition (or [partial] FGFR1 deletion) in HS-5 stromal cells decreases exosome production/secretion and probably alters the content of exosomes. However, the story from the point of view of the leukemic cell lines is largely unexplored and mechanistically vague. Does FGFR1 deletion in the leukemic cell lines change the effectiveness of HS-5 CM? Does PD173074 completely shut off FGFR1 signaling in the leukemic cell lines? Perhaps I am missing something, but I find it surprising that inhibition of FGFR1 (in the leukemic cells) does not at least partially reverse the protective effects of HS-5 CM. This is largely based on the authors' reasonable assertion that the most important exosome cargo is FGF2.

Conditioned media from HS-5 cells contains a number of cytokines and other secreted factors in addition to FGF2-containing exosomes so the impact of adding an FGFR inhibitor such as PD173074 to CM is less pronounced in this setting (Figure 4A). If ECVs are isolated from HS-5 CM and then added to K562 or MOLM14 cells, then the inhibitory effect of PD173074 is more pronounced, although there is still some protection that is not blocked by FGFR inhibition. This is most likely due to protection from other components of exosomes/ECVs. To allow comparison with pure activation of FGFR, we did the same experiment with recombinant FGF2, in which case PD173074 completely blocks the protective effect. The data has been added to Figure 1—figure supplement 1.

The most impressive reduction in protection still comes from treating the HS-5 cells with PD173074 prior to collection of CM (Figure 4A). We found a similar reduction in protection with partial genetic silencing (Figure 6—figure supplement 2) and with genetic deletion of FGF2 from the leukemia microenvironment (Figure 7). This leads to reduced secretion of FGF2 in exosomes and likely has other effects on secretion of protective factors.

3) Several TKIs used in CML have target profiles that include FGFR, most notably the pan-FGFR TKI, ponatinib. Midostaurin, the currently approved frontline TKI for FLT3-mutated AML, inhibits FGFR1 and 2. Would patients treated with these TKIs derive additional benefit from collateral inhibition of FGFR1 in stromal cells (and possibly in leukemic cells)? I expected to see a comparison of imatinib and ponatinib in the study. I also thought this would be worth testing in the mouse model, as mentioned below.

We looked at ponatinib extensively in our previous CML publication (Traer et al. Blood 2014) and argue that additional FGFR inhibition contributes to the effectiveness of ponatinib in non-mutated, resistant CML patients. We have also found that midostaurin blocks many of the protective factors of stroma when MOLM14 cells are co-cultured with HS-5 cells (data not shown), similar to what we have previously published with quizartinib and PD173074 combination treatment (Traer et al. Cancer Research 2016). However, both ponatinib and midostaurin affect a number of additional pathways, especially at higher doses, so we chose to use specific inhibitors of FGFR in our experiments and the mouse model with deletion of FGF2 to get around the issue of off-target effects.

To further address this issue and minor point 1 below, we compared protection with recombinant FGF2, purified HS-5 ECVs, and CM with ECVs removed, using multiple ABL and FLT3 inhibitors. The data is now included in Figure 1—figure supplement 1. As predicted, higher doses of ponatinib and midostaurin are both able to overcome the protective effects of FGF2 and HS-5 ECVs at doses near the reported IC50 for FGFR (between 10-100 nM with ponatinib [Traer et al. Blood 2014] and 200nM midostaurin [Chen et al. PNAS 2014]).

4) Panels 4F,G show data for primary AML stromal cells (n = 42). The legend should specify how many AML patients are represented and how many data points are from each patient. Are some of these FLT3-ITD patients, and if so, can you indicate them as colored dots? This display item and the corresponding Materials and methods, Results and Discussion require more detail. This has potential to be an intriguing part of the story but the presentation is murky. Is it possible to include a comparison to HS-5 and HS-27 in these panels? Are the FGFR1 and FGF2 levels (and their correlations) substantially different from normal bone marrow? Should we expect them to be? Please consider strengthening this part of the manuscript. I could not glean much as presented.

There were nine patients with FLT3-ITD, and these samples are now indicated in Figure 4G, as well as more information about the stromal samples themselves. The primary stromal samples were collected from a variety of de novo and relapsed AML patients and therefore represents a heterogeneous mixture of AML patients at both diagnosis and relapse. There wasn’t a clear association of FGF2 and FGFR1 overexpression with FLT3 ITD, genetic markers, or previous treatment; but there were not enough samples to be conclusive. We did not directly compare expression levels to HS-5 and HS-27 by QPCR.

In our previous publications, the amount of FGF2 in CML and AML marrow core biopsies by immunohistochemistry was not significantly increased compared to normal marrow prior to treatment, and FGF2 only became significantly increased after treatment (Traer et al. Blood 2014, Traer et al. Cancer Research 2016). Therefore we would not necessarily expect more FGF2 expression in stromal samples from patients at diagnosis. We are currently culturing stromal samples from AML patients treated with a FLT3 inhibitor as part of a clinical trial to evaluate change in FGF2 expression over time, but do not have this data yet.

To us, the important feature of the primary stromal expression data is the coordinate upregulation of FGF2 and FGFR1 to drive autocrine activation in subsets of primary stromal samples. This would be predicted by our previous data, as well as published data on FGF2-FGFR1 autocrine activation in murine marrow stromal cells during stress hematopoiesis (Itkin et al., 2012, Zhao et al., 2012). Finding evidence for this autocrine loop in primary cells indicates it can be activated under the correct conditions. We are very interested to discover what signals from treated leukemia cells stimulates FGF2 and FGFR1 expression in marrow stroma, and this is currently under investigation.

The text was changed as follows:

“To evaluate FGF2 and FGFR1 expression in primary leukemia stroma, bone marrow aspirates from a series of leukemia patients were cultured ex vivo and FGF2 and FGFR1-4 expression quantified by RT-PCR (Figure 4F). FGFR1 and FGF2 transcripts were the most highly expressed in primary stroma, and there was a strong positive correlation between FGFR1 and FGF2 expression (Figure 4G, r²=0.5683 and p<0.0001 on nonparametric correlation). This indicates that FGF2 and FGFR1 expression are coordinately regulated in primary marrow stromal cells consistent with activation of an FGF2-FGFR1 autocrine loop. There were 9 stromal cultures from AML patients with FLT3 ITD (indicated with red dots), but most of them were newly diagnosed, and based upon our previous data we would not expect increased expression of FGF2¹⁶. Similar to our observations in cell lines described above, we also detected FGFR1 and FGF2 in ECVs derived from primary marrow stromal cultures (Figure 4—figure supplement 2A). However, primary marrow stromal cells grow slowly and produce smaller amounts of ECVs, so we were unable to evaluate the effect of FGFR inhibitors on cell morphology, growth, and ECV production with primary marrow stromal cells.”

Minor Comments:

1) Figure 1: In panel A, what is the result if the same experiment is carried out with ponatinib (10-25 nM)? dasatinib (10-25 nM)? In panel B, with midostaurin (1 µM)? This comment is somewhat redundant with major point (3) above; here, I am requesting that the data are included, at least in the supplement.

Now included in Figure 1—figure supplement 1. Please see above for detail.

2) Why was 1 µM imatinib used? This concentration, at least at the 48 h timepoint, does not reduce proliferation (Figure 1A) to the extent that AC220 does in MOLM14 cells. Thus, it is difficult to determine the extent to which CM provides a protective effect. What happens if a higher concentration of imatinib is used, or if a more potent TKI is used at a concentration that matches the AC220 magnitude of reduced proliferation?

We used 1 µM imatinib primarily for comparison with our previous experiments. Now included in Figure 1—figure supplement 1 are a variety of ABL inhibitors and concentrations. Even with higher concentrations and more potent inhibitors K562 cells undergo apoptosis more slowly than MOLM14 cells.

3) Regarding the FGFR inhibitors:

– Why two? BGJ-398 is described as 'selective' but I did not find the rationale for needing it in the study (which began with PD173074).

– What is the direct evidence/readout to confirm that these inhibitors are working (and to measure the extent of inhibition)?

Previous reviewers requested experiments with more selective FGFR inhibitors and BGJ-398 is one of the newer and more selective inhibitors.

4) Perhaps it's not a reasonable comparison, but it is surprising that shRNA-based elimination of FGFR1 does not reduce FGF2 levels in HS-5 cells (Figure 6A), but FGFR inhibition does (Figure 5C). Also, the HS-5 CRISPR FGFR1.1 and 1.2 lines show reduced FGF2. The HS-5 CRISPR FGF2.1 and 2.2 lines show enhanced FGFR1 but reduced FGFR1 in ECVs. Can the authors comment on / explain these points?

We suspect most of the variability with FGF2 levels after shRNA or CRISPR/CAS of FGFR1 or FGFR inhibition are assay specific and related to when the cells were evaluated by immunoblot. Reviewer #1 also commented on this, and a more complete discussion is outlined above.

5) In Figure 6B, I would like to be able to evaluate whether reduced FGFR1 brings about a reduction in FGF2, but this data is not provided. In general, both FGF2 and FGFR1 should be shown in immunoblot stacks. Sometimes, it is one or the other.

Reviewer #1 also raised this issue and the absence of FGF2 staining in that figure was a technical issue (see above). Since we often had limited supplies of ECVs, we had to run the entire sample to obtain enough signal for detection, which did not allow for repeat immunoblots.

6) Figure 7:

– why is nilotinib used in 7A? All other data are with imatinib.

– the main point of the paper seems to be that interrupting the FGFR1-FGF2 network in stromal cells would decrease the protective effects emanating from the microenvironment. To test this, shouldn't arms with "PD PD173074" alone, and with "nilotinib + PD173074" be included?

– why did you choose this model rather than a FLT3-ITD model? The jumping back and forth between CML and AML is distracting. Could the authors consider a focus on MOLM14 and FLT3-ITD driven AML in the main manuscript, with the CML work allocated to the supplement?

We used the more potent inhibitor nilotinib to allow once daily gavage. This still allows for disease suppression and reduces trauma to the mice (and lab technicians). Imatinib is usually given twice daily by gavage.

The combination of an FGFR inhibitor and ABL inhibitor would be predicted to also reduce resistance, but the FGF2 -/- is a cleaner genetic model. We are currently trying to find pharma support to test relevant combinations with an FGFR inhibitor in clinical development.

The BCR-ABL model is a well-characterized model in the lab and does not require breeding. The FLT3-ITD and TET2 mouse model that we have access to would require extensive back-breeding with the FGF2 +/+ and -/- mice, adding significantly more time and expense.

We feel the focus of this manuscript on marrow stroma provides a unifying theme for our previous discoveries that FGF2 promotes resistance in both CML and AML. Our data also suggests that targeting the stroma directly with FGFR inhibitors is a novel therapeutic target in multiple leukemias.

7) Please address the following clerical issues:

– abbreviations are defined several times (examples: TKI; conditioned media)

– conventions are not consistently followed (ml vs. mL; h vs. hrs vs. hours; spacing between value and unit [e.g. 10nM vs. 10 nM]; no spacing vs. spacing preceding a citation)

– Chemical formulas lack proper subscripting (possibly an inherent submission portal limitation)

– Please check supplier names (Genscript or GenScript? Is Jackson laboratories the correct name of this supplier?)

– References 14 and 30 are identical

– A few article titles in References section are title case (#4, 15, 16, for example)

– Reference 23 lacks page number, etc. The final manuscript is 2016, not 2015

– Reference 25 lacks journal name, etc.

– Figure 2 legend: NB40 or NP-40?

– Final paragraph of subsection “FGF2 is Contained in Stromal Cell ECVs and Exosomes”: "membrate"

– First paragraph of subsection “FGF2-FGFR1 Signaling Promotes Stromal Growth and Paracrine Protection of Leukemia”: "withg"

– "proteinase K" vs. "Proteinase K"

– "PD173074" vs. "PD-173074"

– Second paragraph of the Discussion: a callout to Figure 3E is made. Should this be 3D (there is no panel 3E)?

Our apologies for the errors and inconsistencies. This has been corrected, although I left a definition of conditioned media in both the text and Figure 1 legend since I like to look at figures and legends first.

Additional data files and statistical comments:

I asked the authors to consider including additional data based on experiments with tyrosine kinase inhibitors beyond imatinib and AC220. If they choose to do this, inclusion of the data files is warranted. I do not see the need for any other additional data files.

The Prism data files were uploaded.

Author details

1. Nathalie Javidi-Sharifi

   Knight Cancer Institute, Oregon Health & Science University, Portland, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Jacqueline Martinez

   Competing interests

   No competing interests declared

2. Jacqueline Martinez

   Knight Cancer Institute, Oregon Health & Science University, Portland, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft

   Contributed equally with

   Nathalie Javidi-Sharifi

   Competing interests

   No competing interests declared

3. Isabel English

   Knight Cancer Institute, Oregon Health & Science University, Portland, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

4. Sunil K Joshi

   Knight Cancer Institute, Oregon Health & Science University, Portland, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

5. Renata Scopim-Ribeiro

   Knight Cancer Institute, Oregon Health & Science University, Portland, United States

   Contribution

   Data curation, Investigation, Visualization

   Competing interests

   No competing interests declared

6. Shelton K Viola

   Knight Cancer Institute, Oregon Health & Science University, Portland, United States

   Present address

   Department of Pediatrics, Division of Pediatric Hematology-Oncology, Naval Medical Center Portsmouth, Portsmouth, United States

   Contribution

   Data curation, Investigation

7. David K Edwards V

   Knight Cancer Institute, Oregon Health & Science University, Portland, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

8. Anupriya Agarwal

   1. Knight Cancer Institute, Oregon Health & Science University, Portland, United States
   2. Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

9. Claudia Lopez

   1. Knight Cancer Institute, Oregon Health & Science University, Portland, United States
   2. Center for Spatial Systems Biomedicine, Oregon Health & Science University, Portland, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

10. Danielle Jorgens

    1. Knight Cancer Institute, Oregon Health & Science University, Portland, United States
    2. Center for Spatial Systems Biomedicine, Oregon Health & Science University, Portland, United States

    Contribution

    Resources, Investigation

    Competing interests

    No competing interests declared

11. Jeffrey W Tyner

    1. Knight Cancer Institute, Oregon Health & Science University, Portland, United States
    2. Department of Cell, Developmental & Cancer Biology, Oregon Health & Science University, Portland, United States

    Contribution

    Resources, Writing—review and editing

    Competing interests

    No competing interests declared

12. Brian J Druker

    1. Knight Cancer Institute, Oregon Health & Science University, Portland, United States
    2. Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, United States
    3. Howard Hughes Medical Institute, Chevy Chase, United States

    Contribution

    Resources, Supervision, Writing—review and editing

    Competing interests

    Is currently principal investigator or co-investigator on Novartis clinical trials. His institution, OHSU, has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not derive salary, nor does his lab receive funds from these contracts.

13. Elie Traer

    1. Knight Cancer Institute, Oregon Health & Science University, Portland, United States
    2. Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    traere@ohsu.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8844-2345

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