Formation of malignant, metastatic small cell lung cancers through overproduction of cMYC protein in TP53 and RB1 depleted pulmonary neuroendocrine cells derived from human embryonic stem cells

Reviewer #1 (Public Review):

Summary:
The authors introduced their previous paper with the concise statement that "the relationships between lineage-specific attributes and genotypic differences of tumors are not understood" (Chen et al., JEM 2019, PMID: 30737256). For example, it is not clear why combined loss of RB1 and TP53 is required for tumorigenesis in SCLC or other aggressive neuroendocrine (NE) cancers, or why the oncogenic mutations in KRAS or EGFR that drive NSCLC tumorigenesis are found so infrequently in SCLC. This is the main question addressed by the previous and current papers.

One approach to this question is to identify a discrete set of genetic/biochemical manipulations that are sufficient to transform non-malignant human cells into SCLC-like tumors. One group reported the transformation of primary human bronchial epithelial cells into NE tumors through a complex lentiviral cocktail involving the inactivation of pRB and p53 and activation of AKT, cMYC, and BCL2 (PARCB) (Park et al., Science 2018, PMID: 30287662). The cocktail previously reported by Chen and colleagues to transform human pluripotent stem-cell (hPSC)-derived lung progenitors (LPs) into NE xenografts was more concise: DAPT to inactivate NOTCH signaling combined with shRNAs against RB1 and TP53. However, the resulting RP xenografts lacked important characteristics of SCLC. Unlike SCLC, these tumors proliferated slowly and did not metastasize, and although small subpopulations expressed MYC or MYCL, none expressed NEUROD1.

MYC is frequently amplified or expressed at high levels in SCLC, and here, the authors have tested whether inducible expression of MYC could increase the resemblance of their hPSC-derived NE tumors to SCLC. These RPM cells (or RPM T58A with stabilized cMYC) engrafted more consistently and grew more rapidly than RP cells, and unlike RP cells, formed liver metastases when injected into the renal capsule. Gene expression analyses revealed that RPM tumor subpopulations expressed NEUROD1, ASCL1, and/or YAP1.

The hPSC-derived RPM model is a major advance over the previous RP model. This may become a powerful tool for understanding SCLC tumorigenesis and progression and for discovering gene dependencies and molecular targets for novel therapies. However, the specific role of cMYC in this model needs to be clarified.

cMYC can drive proliferation, tumorigenesis, or apoptosis in a variety of lineages depending on concurrent mutations. For example, in the Park et al., study, normal human prostate cells could be reprogrammed to form adenocarcinoma-like tumors by activation of cMYC and AKT alone, without manipulation of TP53 or RB1. In their previous manuscript, the authors carefully showed the role of each molecular manipulation in NE tumorigenesis. DAPT was required for NE differentiation of LPs to PNECs, shRB1 was required for expansion of the PNECs, and shTP53 was required for xenograft formation. cMYC expression could influence each of these steps, and importantly, could render some steps dispensable. For example, shRB1 was previously necessary to expand the DAPT-induced PNECs, as neither shTP53 nor activation of KRAS or EGFR had no effect on this population, but perhaps cMYC overexpression could expand PNECs even in the presence of pRB, or even induce LPs to become PNECs without DAPT. Similarly, both shRB1 and shTP53 were necessary for xenograft formation, but maybe not if cMYC is overexpressed. If a molecular hallmark of SCLC, such as loss of RB1 or TP53, has become dispensable with the addition of cMYC, this information is critically important in interpreting this as a model of SCLC tumorigenesis.

To interpret the role of cMYC expression in hPSC-derived RPM tumors, we need to know what this manipulation does without manipulation of pRB, p53, or NOTCH, alone or in combination. Seven relevant combinations should be presented in this manuscript: (1) cMYC alone in LPs, (2) cMYC + DAPT, (3) cMYC + shRB1, (4) cMYC + DAPT + shRB1, (5) cMYC + shTP53, (6) cMYC + DAPT + shTP53, and (7) cMYC + shRB1 + shTP53. Wild-type cMYC is sufficient; further exploration with the T58A mutant would not be necessary.

This reviewer considers that there should be a presentation of the effects of these combinations on LP differentiation to PNECs, expansion of PNECs as well as other lung cells, xenograft formation and histology, and xenograft growth rate and capacity for metastasis. If this could be clarified experimentally, and the results discussed in the context of other similar approaches such as the Park et al., paper, this study would be a major addition to the field.

Reviewer #2 (Public Review):

Summary:
Chen et al use human embryonic stem cells (ESCs) to determine the impact of wildtype MYC and a point mutant stable form of MYC (MYC-T58A) in the transformation of induced pulmonary neuroendocrine cells (PNEC) in the context of RB1/P53 (RP) loss (tumor suppressors that are nearly universally lost in small cell lung cancer (SCLC)). Upon transplant into immune-deficient mice, they find that RP-MYC and RP-MYC-T58A cells grow more rapidly, and are more likely to be metastatic when transplanted into the kidney capsule, than RP controls. Through single-cell RNA sequencing and immunostaining approaches, they find that these RPM tumors and their metastases express NEUROD1, which is a transcription factor whose expression marks a distinct molecular state of SCLC. While MYC is already known to promote aggressive NEUROD1+ SCLC in other models, these data demonstrate its capacity in a human setting that provides a rationale for further use of the ESC-based model going forward. Overall, these findings provide a minor advance over the previous characterization of this ESC-based model of SCLC published in Chen et al, J Exp Med, 2019.

The major conclusion of the paper is generally well supported, but some minor conclusions are inadequate and require important controls and more careful analysis.

Strengths:
1. Both MYC and MYC-T58A yield similar results when RP-MYC and RP-MYCT58A PNEC ESCs are injected subcutaneously, or into the renal capsule, of immune-deficient mice, leading to the conclusion that MYC promotes faster growth and more metastases than RP controls.

2. Consistent with numerous prior studies in mice with a neuroendocrine (NE) cell of origin (Mollaoglu et al, Cancer Cell, 2017; Ireland et al, Cancer Cell, 2020; Olsen et al, Genes Dev, 2021), MYC appears sufficient in the context of RB/P53 loss to induce the NEUROD1 state. Prior studies also show that MYC can convert human ASCL1+ neuroendocrine SCLC cell lines to a NEUROD1 state (Patel et al, Sci Advances, 2021); this study for the first time demonstrates that RB/P53/MYC from a human neuroendocrine cell of origin is sufficient to transform a NE state to aggressive NEUROD1+ SCLC. This finding provides a solid rationale for using the human ESC system to better understand the function of human oncogenes and tumor suppressors from a neuroendocrine origin.

Weaknesses:
1. There is a major concern about the conclusion that MYC "yields a larger neuroendocrine compartment" related to Figures 4C and 4G, which is inadequately supported and likely inaccurate. There is overwhelming published data that while MYC can promote NEUROD1, it also tends to correlate with reduced ASCL1 and reduced NE fate (Mollaoglu et al, Cancer Cell, 2017; Zhang et al, TLCR, 2018; Ireland et al, Cancer Cell, 2020; Patel et al, Sci Advances, 2021). Most importantly, there is a lack of in vivo RP tumor controls to make the proper comparison to judge MYC's impact on neuroendocrine identity. RPM tumors are largely neuroendocrine compared to in vitro conditions, but since RP control tumors (in vivo) are missing, it is impossible to determine whether MYC promotes more or less neuroendocrine fate than RP controls. It is not appropriate to compare RPM tumors to in vitro RP cells when it comes to cell fate. Upon inspection of the sample identity in S1B, the fibroblast and basal-like cells appear to only grow in vitro and are not well represented in vivo; it is, therefore, unclear whether these are transformed or even lack RB/P53 or express MYC. Indeed, a close inspection of Figure S1B shows that RPM tumor cells have little ASCL1 expression, consistent with lower NE fate than expected in control RP tumors.

In addition, since MYC appears to require Notch signaling to induce NE fate (Ireland et al), the presence of DAPT in culture could enrich for NE fate despite MYC's presence. It's important to clarify in the legend of Fig 4A which samples are used in the scRNA-seq data and whether they were derived from in vitro or in vivo conditions (as such, Supplementary Figure S1B should be provided in the main figure). Given their conclusion is confusing and challenges robustly supported data in other models, it is critical to resolve this issue properly. I suspect when properly resolved, MYC actually consistently does reduce NE fate compared to RP controls, even though tumors are still relatively NE compared to completely distinct cellular identities such as fibroblasts.

2. The rigor of the conclusions in Figure 1 would be strengthened by comparing an equivalent number of RP animals in the renal capsule assay, which is n = 6 compared to n = 11-14 in the MYC conditions.

3. Statistical analysis is not provided for Figures 2A-2B, and while the results are compelling, may be strengthened by additional samples due to the variability observed.

4a. Related to Figure 3, primary tumors and liver metastases from RPM or RPM-T58A-expressing cells express NEUROD1 by immunohistochemistry (IHC) but the putative negative controls (RP) are not shown, and there is no assessment of variability from tumor to tumor, ie, this is not quantified across multiple animals.

4b. Relatedly, MYC has been shown to be able to push cells beyond NEUROD1 to a double-negative or YAP1+ state (Mollaoglu et al, Cancer Cell, 2017; Ireland et al, Cancer Cell, 2020), but the authors do not assess subtype markers by IHC. They do show subtype markers by mRNA levels in Fig 4B, and since there is expression of ASCL1, and potentially expression of YAP1 and POU2F3, it would be valuable to examine the protein levels by IHC in control RP vs. RPM samples.

5. Given that MYC has been shown to function distinctly from MYCL in SCLC models, it would have raised the impact and value of the study if MYC was compared to MYCL or MYCL fusions in this context since generally, SCLC expresses a MYC family member. However, it is quite possible that the control RP cells do express MYCL, and as such, it would be useful to show.

Reviewer #3 (Public Review):

Summary:
The authors continue their study of the experimental model of small cell lung cancer (SCLC) they created from human embryonic stem cells (hESCs) using a protocol for differentiating the hESCs into pulmonary lineages followed by NOTCH signaling inactivation with DAPT, and then knockdown of TP53 and RB1 (RP models) with DOX inducible shRNAs. To this published model, they now add DOX-controlled activation of expression of a MYC or T58A MYC transgenes (RPM and RPMT58A models) and study the impact of this on xenograft tumor growth and metastases. Their major findings are that the addition of MYC increased dramatically subcutaneous tumor growth and also the growth of tumors implanted into the renal capsule. In addition, they only found liver and occasional lung metastases with renal capsule implantation. Molecular studies including scRNAseq showed that tumor lines with MYC or T58A MYC led surprisingly to more neuroendocrine differentiation, and (not surprisingly) that MYC expression was most highly correlated with NEUROD1 expression. Of interest, many of the hESCs with RPM/RPMT58A expressed ASCL1. Of note, even in the renal capsule RPM/RPMT58A models only 6/12 and 4/9 mice developed metastases (mainly liver with one lung metastasis) and a few mice of each type did not even develop a renal sub capsule tumor. The authors start their Discussion by concluding: " In this report, we show that the addition of an efficiently expressed transgene encoding normal or mutant human cMYC can convert weakly tumorigenic human PNEC cells, derived from a human ESC line and depleted of tumor suppressors RB1 and TP53, into highly malignant, metastatic SCLC-like cancers after implantation into the renal capsule of immunodeficient mice.".

Strengths:
The in vivo study of a human preclinical model of SCLC demonstrates the important role of c-Myc in the development of a malignant phenotype and metastases. Also the role of c-Myc in selecting for expression of NEUROD1 lineage oncogene expression.

Weaknesses:
There are no data on results from an orthotopic (pulmonary) implantation on generation of metastases; no comparative study of other myc family members (MYCL, MYCN); no indication of analyses of other common metastatic sites found in SCLC (e.g. brain, adrenal gland, lymph nodes, bone marrow); no studies of response to standard platin-etoposide doublet chemotherapy; no data on the status of NEUROD1 and ASCL1 expression in the individual metastatic lesions they identified.

Author Response

We are grateful to the three reviewers and the editors who have provided comments about our manuscript, "Formation of malignant, metastatic small cell lung cancers through overproduction of cMYC protein in TP53 and RB1 depleted pulmonary neuroendocrine cells derived from human embryonic stem cells.”

We are pleased that the reviewers recognized the importance of the problem we have addressed – namely, the need for better models of small cell lung cancer, a relatively common and refractory cancer. We also appreciate their acknowledgement of the significance of our major finding: that addition of an efficiently expressed CMYC transgene to neuroendocrine cells derived from human embryonic stem cells in which the RB1 and TP53 genes have been suppressed serves to drive aggressive growth and metastatic spread, rendering this system an appealing one for future studies of this recalcitrant cancer. Further, we acknowledge that more work needs to be done to more fully characterize and better understand the mechanistic features of this model system and to exploit it for therapeutic purposes.

More specifically, we agree with the reviewers that this manuscript would be stronger if it included: (i) tests of other oncogenes, especially other members of the MYC gene family, to serve as drivers of tumor growth and metastasis and tests of orthotropic implantation of cells into the lung; (ii) descriptions of how such tumors with various genotypes respond to therapeutic approaches, both established and novel; and (iii) a more complete assessment of the contribution of abundant MYC proteins to physiological changes in tumor cells, such as growth, apoptosis, and invasion.

While we wish we could provide such information, it is unrealistic to believe that it will be generated by the current constellation of authors in the foreseeable future. Data in the present manuscript has been generated over nearly five years, mostly in the early phases of that interval. Since then, some of us have moved from one institution to another, and some have shifted the focus of our studies. Further delays in publishing the main messages in this paper will only delay the pursuit of further studies, most likely by others. Indeed, one of the strongest justifications for the novel publication policies at eLife is to return control of the time for dissemination of results to the hands of the authors. Our situation illustrates the wisdom of that approach.

We also note that the reviewers have raised a few issues that we aim to clarify by revisions of the current manuscript, thereby creating an improved Version of Record, within the next few weeks. We acknowledge here the significance of those issues and the ambiguities noted by the reviewers.

The issues include the following point noted by more than one reviewer: our claim that expression of the CMYC oncogene increases the neuroendocrine character of the tumors. We recognize that this observation may be influenced by the nature of the analysis (single cell or bulk RNA sequencing), the choice of lineage markers (eg, NEUROD1 or ASCL1 or others), and the statistical evaluation of the data. We will review these aspects of the problem and make appropriate changes in the text to be submitted as the Version of Record.

Reviewer 1 also makes a good point about the possible effects of CMYC on the differentiation of hESC-derived lung progenitors (LPs). In this paper, we examine this issue only in LPs in which the tumor suppressor genes, RB1 and TP53, have been suppressed. Further studies of the effect of CMYC on differentiation of LPs with various combinations of functional tumor suppressor genes might well prove valuable in exploring the origins of SCLC.

Finally, we wish to note that a topic discussed by Reviewer 1 (and by us) about the still poorly understood relationship between cancer genotypes and cell lineages has been partially addressed in a paper from our group that has been accepted for publication in Science.