Major advances in cancer therapy have improved the treatment options for many patients. However, many cancer treatments are toxic or have severe side effects, making them difficult for patients to tolerate. One cause of these side effects is that many cancer therapies kill both normal cells and cancer cells. Developing cancer therapies that are more targeted is therefore a priority in cancer research.

Acute myeloid leukemia is a type of blood cancer that has proven difficult to treat without causing serious side effects. This cancer is very aggressive and only about 1 in 4 patients are successfully cured of their cancer. At present, physicians treat acute myeloid leukemia with chemotherapy, which kills both the cancer cells and some of the patient's healthy cells.

Many patients with acute myeloid leukemia have mutations in the gene encoding an enzyme called Fms-like tyrosine kinase 3 (FLT3). This mutation makes the enzyme permanently active, and patients with the mutation have a greater risk of their cancer recurring or death. Scientists have recently discovered that treatments that inhibit the FLT3 enzyme can be effective against cancer. However, the drugs investigated so far also interfere with the patient's ability to produce new blood cells, which can lead to infections or an inability to recover from bleeding. Therefore, no new drugs have yet been approved for general use.

Warkentin et al. suspected the reason for the adverse effects of FLT3 inhibitors is that these drugs also inhibit another enzyme necessary for blood cell production. Previous work showed that inhibiting one or the other of the enzymes still allows blood cells to be produced as normal: it is only when both are inhibited that production problems arise. Warkentin et al. therefore looked for a chemical that inhibits only the FLT3 enzyme and found one called Star 27. Tests revealed that this inhibits FLT3 and prevents the growth and spread of cancerous cells but does not impair blood cell production. Additionally, Star 27 continues to work even when mutations arise in the cancer cells that cause resistance to other FLT3 inhibitors.

The findings demonstrate that when it comes to drug development, it is sometimes as important to avoid certain molecular targets as it is to hit others. Understanding the network of enzymes that FLT3 works with could therefore help researchers to develop more effective and safer cancer treatments.

Kinase inhibitors are among the fastest growing new class of therapeutics for treating cancer, with 25 new kinase inhibitors approved by the FDA in the last 14 years (Mullard, 2014). Since these agents almost exclusively target a kinase's highly conserved ATP binding pocket, achieving selectivity is problematic. A limiting feature of kinase inhibitors is that their ability to target multiple wild-type kinases in normal tissues limits the doses that can be used to target the mutant kinase in the tumor tissue. The key question, in light of the complex kinase networks in all cells, is which anti-targets should be avoided, in order to limit toxicity in normal tissues. Our work (Kung et al., 2006; Dar et al., 2012) and that of others (Ahmad and Eisen, 2004; Wilhelm et al., 2006) have highlighted the potent effects of multi-targeted kinase inhibitors, revealing unexpected effects when several kinases are inhibited rather than each one individually (synthetic lethal effects as well as positive epistatic effects). By understanding the synthetic lethal effects on normal cells and developing selective inhibitors which avoid even a small number of ‘off-target’ kinases, we believe that clinical agents with an improved therapeutic index can be developed.

One disease where this anti-target (Dar et al., 2012) concept is particularly needed is AML, a rapidly fatal blood cancer comprising 2% of cancer deaths in the United States in 2013 and a disproportionate number of new cases (ca. 19,000, 30%) and deaths (ca. 10,500, 44%) relative to all leukemias (Leukemia and Lymphoma Society, 2014). FDA-approved chemotherapy has not advanced beyond general cytotoxic agents, and no highly active therapies have been approved in over 30 years. The cure-rate for AML remains approximately 25%. Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase (RTK) regulating hematopoietic differentiation that is mutated in 30–35% of AML cases, making it the most frequently mutated gene in AML (Mizuki et al., 2000). 25% of AML patients present with juxtamembrane (JM) domain duplications termed internal tandem duplications (FLT3-ITD) (Kindler et al., 2005). The ITD mutation is thought to render FLT3 constitutively active by disrupting an autoinhibitory function of the JM domain. Patients with FLT3-ITD mutations have significantly increased relapse rates and shortened survival, thus illustrating a major unmet therapeutic need.

FLT3-ITD was recently validated as a therapeutic target in AML (Smith et al., 2012). Several targeted FLT3 tyrosine kinase inhibitors (TKIs) have been investigated; however, none have advanced beyond Phase III clinical trials (Smith and Shah, 2013). The FLT3 TKI AC220 (Quizartinib) (Chao et al., 2009) achieves substantial reduction in leukemic blasts initially in a high proportion of patients and is the most kinome-wide selective clinical candidate (Zarrinkar et al., 2009). However, in spite of its promising selectivity, which is remarkably confined to inhibition of the Class III/PDGFR family of RTK's, AC220-induced myelosuppression represents a major dose-limiting toxicity (Galanis et al., 2012; Smith and Shah). As a result, while many patients achieve clearance of bone marrow (BM) blasts, most experience incomplete recovery of normal blood counts (CRi) and remain at risk of complications such as life-threatening infection or bleeding.

The Class III family of RTKs (comprising FLT3, KIT, CSF1R, PDGFRα, and PDGFRβ) are important regulators of normal hematopoiesis. In 1995, Lemischka and coworkers showed that mice lacking function of either Flt3 or Kit maintained overall normal populations (Mackarehtschian et al., 1995). However, mice lacking both Flt3 and Kit function had a dramatic reduction of hematopoietic cell numbers, ca. 15-fold white cell depletion, reduction of lymphoid progenitors, and postnatal lethality. We propose that KIT is an anti-target (Dar et al., 2012) in the context of pharmacologic inhibition of FLT3. Thus, normal mature hematopoietic populations can be maintained in the context of either Flt3 or Kit inhibition alone but not dual Flt3/Kit inhibition (Bershtein et al., 2006).

This synthetic lethal toxicity relationship between FLT3 and KIT for maintaining normal hematopoietic populations may explain the adverse side effects of the current kinase targeted drugs in clinical development. In a recent single agent Phase II trial, PKC412 failed to achieve a single complete remission (CR). When combined with cytotoxic agents PKC412 showed some promise, achieving a 25% CR rate, but responses were primarily incomplete recovery of peripheral blood counts (CRi, 20%) with over 90% of patients developing grade 3/4 myelosuppression (Strati et al., 2014). While AC220 monotherapy impressively demonstrated a 50% CR rate in a Phase II trial, these consisted primarily of CRi (45%) with few real CRs with complete recovery of blood counts (Cortes et al., 2013), correlating with the similar potency of these agents for both FLT3 and KIT. A recent study showed increased selectivity of the clinical agent crenolanib for FLT3 over KIT and reinforced the correlation between target inhibition, and anti-target avoidance (Dar et al., 2012), which lead to lowered toxicity towards normal hematopoiesis (Galanis et al., 2014). However, the potency of crenolanib for KIT remains too high (IC₅₀ = 67 nM for p-KIT inhibition in TF-1 cells; 65% inhibition at 100 nM, in vitro) (Galanis et al., 2014). This is likely insufficient to fully minimize clinically relevant myelosuppression, as a recent interim analysis reported only a 17% (3/18 patients) composite CR rate in AML patients, with 2/3 of these responders achieving only CRi (Collins et al., 2014). These findings highlight the need for new clinical candidates that better minimize KIT and other Class III RTK inhibition.

While avoiding inhibition of the presumed anti-target, KIT, is one chemical challenge toward inhibitor design, the emergence of on-target resistance is another clinical challenge. We (Smith et al., 2012) and others (Wodicka et al., 2010) have identified the acquisition of secondary FLT3 kinase domain (KD) mutations that cause drug resistance as another limitation of current clinically active FLT3 inhibitors. Mutations at the activation loop residue D835 are particularly clinically problematic. These mutations are proposed to bias the kinase toward the constitutively active conformation by disrupting a hydrogen bond from D835 to S838, and thus limit the efficacy of Type II inhibitors such as AC220. We have recently proposed that a Type I inhibitor, which binds to the active kinase conformation, would circumvent these mutations that confer resistance to AC220 (Smith et al., 2012). New small molecule therapies have been reported to bypass these particular mutations, including crenolanib (Galanis et al., 2014), a Type I inhibitor (Lee et al., 2014; Smith et al., 2014), but the CR rate of crenolanib remains modest (Collins et al., 2014). Moreover, it is likely that a repertoire of drugs will be necessary to combat emerging resistance.

We propose herein a solution to the FLT3/KIT selectivity problem designed to avoid myelosuppression and also retain potency against drug-resistant mutations. The staurosporine scaffold has been utilized pharmacologically for 30 years, and staurosporine analogs have been proven to be potent FLT3 inhibitors (PKC412, CEP701) (Strati et al., 2014), though clinical activity of these compounds has been modest, perhaps caused by lack of potent FLT3 inhibition due to dose-limiting toxicity in vivo. The lactam ring C7 position remains virtually unexplored for modulating selectivity (Wood et al., 1999; Bishop et al., 2000; Heidel et al., 2005). We recently reported that C7-substituted staurosporine analogs, we term ‘staralogs’, are potent and selective inhibitors of engineered analog-sensitive (AS) kinases (Lopez et al., 2013). For example, when C7 (R₁) equals isobutyl (Star 12), AS Src kinase is potently inhibited but WT kinases remain unaffected. However, we also observed that Star 12, in a panel of 319 kinases, weakly inhibits only one WT kinase, FLT3 (57% inhibition at 1 µM; KIT, CSF1R, PDGFRα/β all inhibited <10%). Thus, the C7-alkyl group of Star 12 may allow for weak but selective inhibition of FLT3 over the anti-target KIT, which contributes to myelosuppression when FLT3 is also inhibited. Although substitution of an isobutyl group at C7 reduced potency, we hypothesized that combining the FLT3/KIT selectivity of Star 12 and the potency features of PKC412 would generate an optimal FLT3 inhibitor also capable of targeting emerging mutations (Figure 1A).

Figure 1

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The aggressive nature of FLT3-driven AML and the lack of effective and well-tolerated targeted therapy represent a major unmet therapeutic need. Currently, the most promising clinical targeting agents cause myelosuppression in most patients regardless of remission status. As kinase inhibitor-based therapies evolve, refined selectivity against particular off-targets (so-called anti-targets [Dar et al., 2012]) will drive chemical design (Ciceri et al., 2014). Avoiding specific anti-targets will hopefully lead to a reduction in dose-limiting toxicities that will allow more complete inhibition of the oncogenic drivers of disease.

A former targeted clinical candidate, tandutinib (MLN518), may further demonstrate directly the effects of the FLT3/KIT synthetic lethal toxicity model (Griswold et al., 2004). MLN518 was indicated to have similar IC₅₀s for FLT3 and KIT phosphorylation (220 and 170 nM, respectively). While MLN518 showed promise with reducing FLT3-ITD+ blasts in primary patient-derived samples, exposure of the drug to the BM of healthy individuals at a dose necessary for primary FLT3-ITD+ CFU reduction, led to severe depletion of healthy hematopoietic colony formation at 1 µM.

The most direct evidence exists for FLT3/KIT as a synthetic lethal toxic pair, however, other Class III RTKs may exhibit synthetic lethal effects in terms of dose-limiting toxicity when treating FLT3-driven disease. Another Class III RTK, CSF1R, has been shown via the knockout ‘toothless’ rat model (tl = csf1r^−/−) to be important to osteoclastogenesis (Chen et al., 2011), colon development (Huynh et al., 2013), and for normal macrophage and dendritic cell (DC) production and maintenance (Stanley et al., 1997). Perhaps most importantly, tl rats have been reported with a 32% platelet loss, and this thrombocytopenia was not reversed by BM transplant (Thiede et al., 1996). Star 27 exhibits a large selectivity window with a >16,500/1 CSF1R/FLT3 IC₅₀ ratio (Figure 1C), which may provide a clinical benefit. However, more work is needed to determine the synthetic lethal toxicity of CSF1R since other reports suggest an opposite and more complicated role for it in hematopoiesis (Dai et al., 2002).

Our focus on the staurasporine scaffold maintains the beneficial aspects of PKC412's potency against drug-resistant FLT3 mutations (Figure 6A). Since both staralogs are Type I inhibitors, they are not expected to suffer from the Type II-resistance model at the GK (F691L) and especially activation loop (D835) mutations observed in the clinic (Smith et al., 2012). The reduced potency of Star 27 against FLT3 relative to PKC412 in Ba/F3 cells (Figure 6A) is mitigated by the more relevant comparison in human cells (Figure 2A) as well as the predicted increase in maximum tolerated dosing for clinical candidates avoiding the KIT anti-target. Robustness against emergent mutations is a desirable feature of PKC412 but its pan-Class III RTK inhibition (especially KIT) is likely a major limitation addressed here. Remarkably, the addition of a single methyl group (CH₃) likely caused the observed decrease in toxicity.

The strategy reported here for gaining selectivity for FLT3 inhibition over KIT and other Class III RTKs may have importance in other hematopoietic pathology as well. FLT3 has been shown to be important for classical DC maintenance and necessary for inflammatory DCs (Ramos et al., 2014). Consequently, FLT3 has been proposed to be a drug target for autoimmunity and inflammation, particularly atherosclerosis. The staralog, CEP701, has been investigated in this context but it is expected that its promiscuity, particularly among KIT and CSF1R, will hamper clinical development (Dai et al., 2002). Star 27 should enable a reduction in FLT3-associated inflammation and autoimmunity while allowing KIT and CSF1R to compensate with classical DC maintenance. Furthermore, the generality of our electrostatic Phe GK inhibition coupled with Thr GK avoidance model (Figures 1D, 5) may guide future medicinal chemistry efforts, while our in vivo study shows any promiscuity seen in Figure 1E does not compromise Star 27's efficaciousness in a well-validated in vivo model of hematopoiesis (Davidson and Zon, 2004; de Jong and Zon, 2005; He et al., 2014).

Recent clinical experience with targeted inhibition of mutated kinases in cancer suggests that efficacy is driven by maximal pathway inhibition. In the case of vemurafenib, <80% pathway inhibition in BRAF (V600E) mutant melanoma afforded no tumor shrinkage, while >90% inhibition showed profound clinical benefit (Bollag et al., 2010). The challenge is that inhibitors of the oncogene also exhibit off-target effects on closely related kinases that inevitably lead to dose limiting toxicities. The goal of advancing improved molecular-targeted therapies is to identify the key anti-targets that drive toxicity and develop agents that avoid these effects. Our work highlights the synthetic lethal toxicity of an anti-target (KIT) when the oncogenic kinase (FLT3) is inhibited. As more complete understanding of kinase networks emerge it may be possible to delineate the anti-targets to avoid in numerous disease settings to allow improved kinase inhibitor design that can lead to greater ability to inhibit the disease causing pathway activation while avoiding systemic toxicities.

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Author details

1. Alexander A Warkentin

   Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   AAW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

2. Michael S Lopez

   Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   MSL, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

3. Elisabeth A Lasater

   Division of Hematology and Oncology, University of California, San Francisco, San Francisco, United States

   Contribution

   EAL, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Kimberly Lin

   Division of Hematology and Oncology, University of California, San Francisco, San Francisco, United States

   Contribution

   KL, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Bai-Liang He

   Division of Haematology, Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong

   Contribution

   B-LH, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Anskar YH Leung

   Division of Haematology, Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong

   Contribution

   AYHL, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Catherine C Smith

   Division of Hematology and Oncology, University of California, San Francisco, San Francisco, United States

   Contribution

   CCS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   csmith@medicine.ucsf.edu

   Competing interests

   The authors declare that no competing interests exist.

8. Neil P Shah

   Division of Hematology and Oncology, University of California, San Francisco, San Francisco, United States

   Contribution

   NPS, Drafting or revising the article

   For correspondence

   nshah@medicine.ucsf.edu

   Competing interests

   The authors declare that no competing interests exist.

9. Kevan M Shokat

   Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   KMS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   Kevan.Shokat@ucsf.edu

   Competing interests

   The authors declare that no competing interests exist.

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