Jing Shan Lim

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

From Fly Wings to Targeted Cancer Therapies: A Centennial for Notch Signaling

Since Notch phenotypes in Drosophila melanogaster were first identified 100 years ago, Notch signaling has been extensively characterized as a regulator of cell-fate decisions in a variety of organisms and tissues. However, in the past 20 years, accumulating evidence has linked alterations in the Notch pathway to tumorigenesis. In this review, we discuss the protumorigenic and tumor-suppressive functions of Notch signaling, and dissect the molecular mechanisms that underlie these functions in hematopoietic cancers and solid tumors. Finally, we link these mechanisms and observations to possible therapeutic strategies targeting the Notch pathway in human cancers.

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer

The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10–50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.

Tumor heterogeneity in small cell lung cancer defined and investigated in pre-clinical mouse models

Small cell lung carcinoma (SCLC) is a fast-growing, highly metastatic form of lung cancer. A major difference between SCLC and other forms of lung cancer is that SCLC tumors often respond well to chemotherapy initially; unfortunately, resistant tumors rapidly recur. In addition, despite a large number of clinical trials with a variety of therapeutic agents, little progress has been achieved in the past three decades in improving the survival of SCLC patients. These clinical observations indicate that SCLC tumors have a high degree of plasticity and rapid adaptability to changes in growth conditions. Here we consider recent evidence pointing to several levels of heterogeneity in SCLC that may explain the ability of these tumors to adjust to different microenvironment and therapeutics. In particular, we review new data pointing to the existence of several subpopulations of tumor cells that interact with each other to promote tumor growth. We also discuss how SCLC tumors that look similar at the histopathological level may actually represent distinct subtypes of tumors and how these differences impact the response to specific therapeutic agents. A better understanding of genetic and cellular heterogeneity will guide the development of personalized approaches to help SCLC patients.

Intertumoral Heterogeneity in SCLC Is Influenced by the Cell Type of Origin

The extent to which early events shape tumor evolution is largely uncharacterized, even though a better understanding of these early events may help identify key vulnerabilities in advanced tumors. Here, using genetically defined mouse models of small cell lung cancer (SCLC), we uncovered distinct metastatic programs attributable to the cell type of origin. In one model, tumors gain metastatic ability through amplification of the transcription factor NFIB and a widespread increase in chromatin accessibility, whereas in the other model, tumors become metastatic in the absence of NFIB-driven chromatin alterations. Gene-expression and chromatin accessibility analyses identify distinct mechanisms as well as markers predictive of metastatic progression in both groups. Underlying the difference between the two programs was the cell type of origin of the tumors, with NFIB-independent metastases arising from mature neuroendocrine cells. Our findings underscore the importance of the identity of cell type of origin in influencing tumor evolution and metastatic mechanisms.Significance: We show that SCLC can arise from different cell types of origin, which profoundly influences the eventual genetic and epigenetic changes that enable metastatic progression. Understanding intertumoral heterogeneity in SCLC, and across cancer types, may illuminate mechanisms of tumor progression and uncover how the cell type of origin affects tumor evolution. Cancer Discov; 8(10); 1316-31. ©2018 AACR. See related commentary by Pozo et al., p. 1216 This article is highlighted in the In This Issue feature, p. 1195.

Abstract A37: Tumor heterogeneity in SCLC: A role for endogenous Notch signaling

Small cell lung cancer (SCLC), a very aggressive neuroendocrine (NE) tumor of the lung, constitutes approximately 15% of all lung cancers and has a 5-year survival rate of only 5%. Resistance following initial chemotherapy and radiation treatments accounts for the high rate of recurrence and the overall poor prognosis of SCLC. Through genomic sequencing of 100 human SCLC tumors, the group of Roman Thomas and Martin Peifer identified universal inactivation of RB and p53 and inactivating mutations in the NOTCH receptors in ~25% of the tumors. Accordingly, we found that activation of the Notch pathway in a pre-clinical mouse model for SCLC dramatically reduces the number of tumors and extends the survival of the mice. Acute Notch activation also inhibits NE gene expression and proliferation in SCLC tumor cells, providing further evidence for a tumor suppressive role for Notch in SCLC (George, Lim, et al., Nature, 2015). Intriguingly, however, we recently identified a population of cells in mouse and human SCLC tumors that exhibit endogenous activation of the Notch pathway. This observation raises the question of why tumor cells would activate a tumor suppressive pathway. Consistent with the inhibitory role of the pathway in NE differentiation, these Notch-active SCLC cells comprise a slow-cycling, non-NE compartment within SCLC tumors. Lineage tracing experiments indicate that NE tumor cells generate these non-NE Notch-active cells during tumorigenesis. Importantly, our data suggest that these slow-growing Notch-active SCLC cells may function as tumor-derived stromal-like cells that confer a growth or protective advantage to the NE tumor cells under certain growth conditions. In conclusion, Notch is a potent suppressor of SCLC. However, Notch may have pro-tumorigenic effects in SCLC, possibly under stress conditions. A better understanding of the molecular and cellular roles of Notch is required before therapeutic strategies manipulating the Notch pathway can be used in SCLC patients. Citation Format: Jing Shan Lim, Alvaro Ibaseta, Dian Yang, Nadine S. Jahchan, Julien Sage. Tumor heterogeneity in SCLC: A role for endogenous Notch signaling. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A37.

Abstract LB-168: Comprehensive genomic characterization of small cell lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Small cell lung cancer (SCLC) is a highly aggressive, neuroendocrine tumor of the lung that accounts for 15-20% of all lung cancer malignancies. SCLC patients are usually diagnosed with an extensive stage of a tumor, which complicates further surgical resections. The current standard of care is a platinum-based chemotherapy and targeted therapies have not yet been identified for the treatment of SCLC patients. We sought to comprehensively characterize the genomic alterations in SCLC to identify novel candidate targets for therapy. To this end, we performed whole genome sequencing on 110 tumor-normal pairs and transcriptome sequencing on 80 primary tumors. Following the paradigm of classical Knudson tumor suppressors, TP53 and RB1 were detected with somatic, bi-allelic genomic alterations, thus emphasizing the loss of both tumor suppressors as an obligatory event in SCLC. Two cases did not carry genomic alterations in TP53 and RB1, but instead were found to undergo chromothripsis; genomic rearrangements between chromosome 3 and 11 led to the upregulation of Cyclin D1, thus revealing an alternative mechanism of Rb1 deregulation. Additionally, SCLC tumors were found to harbor complex genomic rearrangements of the RB1 and TP53 family members RBL1 (p107), RBL2 (p130) and TP73, the latter resulting in the oncogenic splice variant TP73Δex2/3. SCLC tumors revealed focal amplifications of MYC transcription factors and FGFR1 in 10% and 6% of the cases analyzed, respectively. Additionally, low frequent alterations were detected in SCLC, such as focal amplifications of IRS2, a candidate oncogene involved in the IGF1R tyrosine kinase signaling pathway. A few SCLC cases were found to harbor mutations with possible immediate therapeutic implications; including mutations in BRAF, KIT and PIK3CA. The integrative study for significantly mutated genes confirmed that the histone acetyl transferases CREBBP and EP300 were inactivated in 25% of the tumors(1). Additionally, SCLC tumors showed frequent alterations of centrosomal proteins and in 25% of the cases inactivating mutations of NOTCH family genes. The transcriptional profile of SCLC tumors confirmed a high expression of neuroendocrine markers and of the NOTCH pathway regulator Dlk1, pointing to the notion that NOTCH functions as a tumor suppressor in SCLC tumors. We sought to analyze the impact of Notch re-activation in a preclinical SCLC mouse model by introducing the Notch intracellular domain (NICD) in mice with conditional lung epithelial triple knock-out (TKO) of Trp53, Rb1 and Rbl2(2). NICD expression dramatically reduced the number of tumors arising in TKO mice and led to an extended survival. Additionally, Notch activation abrogated the expression of neuroendocrine markers. In summary, this whole genome sequencing analysis provides a comprehensive understanding of the genomic nature of SCLC tumors endorsing universal bi-allelic loss of TP53 and RB1 as key events in the pathogenesis of SCLC. This study further emphasizes NOTCH as a tumor suppressor and regulator of neuroendocrine differentiation in SCLC tumors. Furthermore, this large-scale study uncovers several biological key processes and candidate therapeutic targets in this deadly form of cancer. (1) Peifer, M. et al. Nat. Genet. 44, 1104-10 (2012). (2) Schaffer, B. E. et al. Cancer Res. 70, 3877-83 (2010). Citation Format: Julie George, Jing Shan Lim, Martin Peifer, Julien Sage, Roman Thomas. Comprehensive genomic characterization of small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-168. doi:10.1158/1538-7445.AM2015-LB-168