Supplementary Methods from Interaction between MYC and MCL1 in the Genesis and Outcome of Non–Small-Cell Lung Cancer

Supplementary Table 1 from Interaction between MYC and MCL1 in the Genesis and Outcome of Non–Small-Cell Lung Cancer

Supplementary Figure Legends 1-6 from Interaction between MYC and MCL1 in the Genesis and Outcome of Non–Small-Cell Lung Cancer

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC.

RNA-directed DNA synthesis by detergent-disrupted virions of Rous sarcoma virus (RSV) initiates by the covalent attachment of pdA to the 3'-terminal rA of a 4S RNA hydrogen-bonded to the 70S RNA template. This 4S "primer" has structural features of tRNA and can be aminoacylated with methionine. Synthesis and integration of provirus DNA can be monitored in both permissive (duck) and nonpermissive (mouse) cells acutely infected with RSV. The results of these studies, as well as data obtained with RSV-infected mammalian cells which have reverted from a transformed to a pheno-typically normal state, indicate that integration of viral genes into the host chromosome is not sufficient cause for transformation. Pertinent features of virus-specific RNA-directed DNA synthesis in vitro and in vivo are reviewed and compared.

Abstract Overexpression of the MYC proto-oncogene is a common genetic malfunction in various human cancers. This makes the gene product of MYC (Myc) a potentially attractive target for therapeutics. Several issues, however, undermine that attractiveness. First, the tumorigenic culprit is usually a normal allele of MYC. There is no distinguishing feature that might facilitate specificity in therapeutic targeting. Second, MYC is a vital gene in most cells. Therapeutic reduction of its function may require exacting titration. Third, “drugging” of Myc has proven to be difficult and has yet to be reduced to practice. There are at least two ways to attack the overexpression of MYC without directly targeting its function. First, Myc represents a nexus in multiple signaling pathways, making its effect vulnerable to inhibition of functions either upstream or downstream of the nexus. For example, there is some promise in inhibiting a bromodomain protein that facilitates expression of MYC, although this approach may still require careful titration of the inhibition. Second, it is possible to exploit the overexpression of Myc by utilizing synthetic lethal interactions with the inhibition of other gene products. This approach may circumvent the several limitations posed by Myc as a therapeutic target. We have explored three distinctive synthetic lethal interactions with the overexpression of Myc, utilizing both cell lines in vitro and mouse models in which a MYC transgene drives tumorigenesis. 1.) Inhibition of the cdk1 cell cycle kinase, which is reversible with normal cells, but elicits vigorous apoptosis in cells that overexpress Myc. The efficacy of this in preclinical studies has led to a Phase 1b trial with triple-negative breast cancer, in which MYC is commonly overexpressed. 2.) Inhibition of the Aurora B kinase, which is also reversible in normal cells, but kills cells that overexpress Myc by means of both apoptosis and autophagy. We have examined the resistance to this therapy that eventually emerges in a MYC-driven liver cancer. As expected, Aurora B kinase has become resistant to the drug in vivo. But there is also a surprising reduction in nuclear Myc, although the tumors remain dependent upon Myc function. 3.) Inhibition of glutaminase. We have found that overexpression of MYC can reconfigure intermediary metabolism in a way that makes at least some forms of tumors exquisitely dependent upon glutamine. In this setting, deprivation of glutamine by inhibition of glutaminase elicits a strong synthetic lethal interaction not displayed by normal cells. It is widely suspected that cancer stem cells possess inherent chemoresistance that allows them to replenish tumors after they have been diminished by chemotherapy. We have identified a small subpopulation of cells in a Myc-driven liver cancer that are enriched for tumor-initiating capacity and display chemoresistance implemented by the MDR1 transporter. This subpopulation can continuously generate “mature” tumor cells, which no longer express MDR1 and have lost the drug resistance. When utilized early in the course of tumorigenesis, the synthetic lethal interaction with Myc elicited by inhibition of Aurora B kinase can eradicate all evidence of tumor-initiating capacity. Our results with MYC exemplify the potential utility of synthetic lethality as a general approach to cancer therapy. It offers a means to attack otherwise recalcitrant targets (including loss of function by tumor suppressor genes), it can substantially diversify the attack on a particular genetic malfunction, and it may have exceptional utility for the eradication of cancer stem cells. Note: This abstract was not presented at the conference. Citation Format: J. Michael Bishop. MYC as therapeutic target. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA25.

Genomic analysis of human hepatocellular carcinoma (HCC) is potentially confounded by the differentiation state of the hepatic cell-of-origin. Here we integrated genomic analysis of mouse HCC (with defined cell-of-origin) along with normal development. We found a major shift in expression of Wnt and RXR-α pathway genes (up and down, respectively) coincident with the transition from hepatoblasts to hepatocytes. A combined Wnt and RXR-α gene signature categorized HCCs into two subtypes (high Wnt, low RXR-α and low Wnt, high RXR-α), which matched cell-of-origin in mouse models and the differentiation state of human HCC. Suppression of RXR-α levels in hepatocytes increased Wnt signaling and enhanced tumorigenicity, whereas ligand activation of RXR-α achieved the opposite. These results corroborate that there are two main HCC subtypes that correspond to the degree of hepatocyte differentation and that RXR-α, in part via Wnt signaling, plays a key functional role in the hepatocyte-like subtype and potentially could serve as a selective therapeutic target.

BACKGROUND & AIMS:The leukocyte composition of tumors is heterogeneous, as is the involvement of each leukocyte subset in promoting or restraining tumorigenesis. This heterogeneity reflects the tissue of origin, tumor stage, and the functional state of leukocyte activation, but its biological roots remain poorly understood. Since tumorigenesis is driven by various genetic events, we assessed the role of driver genes in shaping the profiles and the roles of leukocytes in tumorigenesis. METHODS:Mouse liver tumors were induced by hepatic overexpression of either MYC or the combination of myristoylated AKT and NRAS(V12) oncogenes via hydrodynamic transfection. A comparative, flow cytometry- and histology-based immunophenotyping of liver-infiltrating leukocytes was performed at various stages of liver tumorigenesis. The roles of the most abundant leukocyte subsets in tumorigenesis were addressed by immunodepletion. The contribution of liver injury was assessed by comparing the injury-inducing hydrodynamic transfection model to a model in which MYC is an inducible transgene. RESULTS:Myristoylated AKT and NRAS(V12) promoted a marked recruitment of CD11b(+)Ly6G(hi)Ly6C(int) neutrophils and CD11b(+)Ly6G(-)Ly6C(hi) monocytes to the liver, but their immunodepletion did not alter tumorigenesis. In contrast, despite minimal invasion by monocytes/neutrophils during MYC-driven tumorigenesis, immunodepletion of these cells reduced MYC tumor burden and extended survival. MYC-driven tumor initiation was augmented specifically by Ly6C+ monocytes and their ability to promote liver injury. CONCLUSIONS:Our results demonstrate that leukocyte profiles do not necessarily predict their involvement in tumorigenesis, the functional role of leukocytes can be shaped by oncogenes, and that monocyte-dependent tissue injury selectively cooperates with MYC during tumorigenesis.

Abstract Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify upstream pathways that regulate Sox9 expression in lung cancer, as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known to induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling. Sox9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse Sox9 promoter are not conserved in humans. We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is downstream of Notch1 in lung adenocarcinoma cell lines. By ChIP we determined that Sox9 is a direct target of Notch1 and using luciferase reporter assays, we located the previously unidentified human RBP-Jκ binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the Sox9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Loss of Notch1 expression led to an induced MET phenotype, characterized by decreased cell invasion/migration, MET-like morphological changes, and increased E-cadherin expression, which were rescued by Sox9 overexpression. Our data also demonstrate that Sox9 contributes to Notch1-induced EMT in lung adenocarcinoma. These results establish Sox9 as a key Notch1 target gene mediating Notch1-induced EMT independent of TGF-β, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen M. Capaccione, Xuehui Hong, Katherine M. Morgan, Thaddeus D. Allen, Gregory D. Miles, Elke K. Markert, J. Michael Bishop, Sharon R. Pine. The role of the novel Notch1-Sox9 signaling axis in NSCLC progression and EMT. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1149. doi:10.1158/1538-7445.AM2014-1149

Sox9 has gained increasing importance both functionally and as a prognostic factor in cancer.We demonstrate a functional role for Sox9 in inducing a mesenchymal phenotype in lung ADC.We show that Sox9 mRNA and protein are overexpressed in lung ADC, particularly those with KRAS mutations.Sox9 expression correlated with the Notch target gene Hes1, and numerous other Notch pathway components.We observed that Sox9 is a potent inducer of lung cancer cell motility and invasion, and a negative regulator of E-cadherin, a key protein that is lost during epithelialmesenchymal transition (EMT).Moreover, we show that Notch1 signaling directly regulates Sox9 expression through a SOX9 promoter binding site, independently of the TGF-β pathway, and that Sox9 participates in Notch-1 induced cell motility, cell invasion, and loss of E-cadherin expression.Together, the results identify a new functional role for a Notch1-Sox9 signaling axis in lung ADC that may explain the correlation of Sox9 with tumor progression, higher tumor grade, and poor lung cancer survival.In addition to Notch and TGF-β, Sox9 also acts downstream of NF-κB and Wnt/β-catenin signaling.Thus, Sox9 could potentially act as a hub to mediate cross-talk among key oncogenic pathways in lung ADC.Targeting Sox9 expression or transcriptional activity could potentially reduce resistance to targeted therapy for lung ADC caused by pathway redundancy.

Abstract Background: Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify the upstream pathways that regulate Sox9 expression in lung cancer as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling pathways. SOX9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse SOX9 promoter are not conserved in humans. Results: We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. We validated the data by identifying significant overlap in Sox9 and Hes1 protein expression levels in a human lung cancer tissue microarray. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is immediately downstream of Notch1, but not Notch3, in lung adenocarcinoma cell lines. By ChIP and luciferase reporter assays, we located the previously unidentified human RBPjk binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the SOX9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling, that TGF-β upregulates both Sox9 and Notch1 expression, and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Sox9 overexpression led to an induced EMT phenotype, characterized by increased cell invasion/migration, and EMT-related gene expression changes. Sox9 mRNA expression correlated with EMT-like gene expression signatures in several gene expression microarray datasets, and Sox9 protein levels were negatively correlated with expression of the epithelial marker, E-cadherin, in a lung cancer tissue microarray. Our data also demonstrate that Notch1-induced EMT in lung adenocarcinoma is mediated, at least partly, through Sox9. Conclusion: These results establish Sox9 as a key Notch1 target gene mediating Notch1- and TGF-β- induced EMT, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen Capaccione, Xuehui Hong, Katherine M. Morgan, Wenyu Liu, Thaddeus Allen, J. Michael Bishop, Sharon R. Pine. Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B17.

Abstract The composition of tumor-associated leukocytes and their pro- or anti-tumor polarization are heterogeneous among different tumor types. In addition, the abundance of leukocyte subtypes can vary even among tumors of the same type. Understanding what determines this heterogeneity is important since the immune cell contexture of tumors can serve as a strong predictor of clinical outcomes and response to anti-cancer therapy. Given that tumorigenesis is driven by mutations in various types of proto-oncogenes and tumor-suppressor genes, we postulated that the identity of driver mutation could shape the leukocytic composition of tumors. To test this, we used the established mouse models for liver tumorigenesis induced by hydrodynamic transfection of c-MYC (MYC) or myr-AKT1+N-RasV12 (AKT+RAS) oncogenes. The resulting somatic integration and long-term overexpression of MYC or AKT+RAS in the liver leads to development of hepatoblastoma and hepatocellular carcinoma, respectively. We profiled liver-infiltrated CD45+ leukocytes in these two models by staining liver single-cell suspensions with lineage-specific leukocyte markers, followed by polychromatic FACS analysis. A massive increase in the CD11b+/F4/80-/Ly6Ghi/Ly6Cmed fraction of CD45+ leukocytes was detected in the AKT+RAS, but not in the MYC model. Compared to the vehicle-injected controls, AKT+RAS livers exhibited a 3-fold increase in this leukocyte population both in pre-neoplastic stages and in tumors. Overexpression of either oncogenes alone did not trigger such increase, suggesting that CD11b+/F4/80-/Ly6Ghi/Ly6Cmed response resulted from cooperation between AKT and RAS. Consistent with the FACS results, IHC staining of liver tissue sections with the neutrophil marker 7/4 antigen revealed an abundant positive staining both in pre-neoplastic livers and in tumors in the AKT+RAS model, while only a small infiltration of 7/4 antigen-positive cells was observed the MYC model only at tumor stage. To address if CD11b+/F4/80-/Ly6Ghi/Ly6Cmed play a role in tumorigenesis, we treated mice with the rat monoclonal Gr-1 antibody to systemically deplete Ly6G/Ly6C-expressing cells. Treatment with Gr-1 starting on day -1 before oncogene transfection ablated CD11b+/F4/80-/Ly6Ghi/Ly6Cmed cells from the livers and showed a tendency to increase the tumor burden in AKT+RAS mice for nearly 50%, suggesting that these cells act in early stages to suppress AKT+RAS-induced tumorigenesis. In contrast, Gr-1 treatment starting on day 4 after oncogene transfection no longer had an effect on AKT+RAS-induced tumorigenesis. Nevertheless, the same protocol significantly extended survival of mice and reduced tumor burden in the MYC model, suggesting a tumor-promoting role of CD11b+/F4/80-/Ly6Ghi/Ly6Cmed in MYC-induced tumorigenesis. In conclusion, our results demonstrate that the leukocytic composition and the nature of immune responses during tumorigenesis are shaped, at least in part, by tumor-driving mutations. We are currently elucidating the mechanism by which CD11b+/F4/80-/Ly6Ghi/Ly6Cmed cells promote or suppress tumorigenesis driven by MYC and AKT+RAS. This abstract is also presented as Poster A46. Citation Format: Vladislava Juric, Brian Ruffell, J. Michael Bishop. Oncogenic driver mutations shape the nature of immune responses in tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR1.

Overexpression of MYC transforms cells in culture, elicits malignant tumours in experimental animals and is found in many human tumours. We now report the paradoxical finding that this powerful oncogene can also act as a suppressor of cell motility, invasiveness and metastasis. Overexpression of MYC stimulated proliferation of breast cancer cells both in culture and in vivo as expected, but inhibited motility and invasiveness in culture, and lung and liver metastases in xenografted tumours. We show further that MYC represses transcription of both subunits of αvβ3 integrin, and that exogenous expression of β3 integrin in human breast cancer cells that do not express this integrin rescues invasiveness and migration when MYC is downregulated. These data uncover an unexpected function of MYC, provide an explanation for the hitherto puzzling literature on the relationship between MYC and metastasis, and reveal a variable that could influence the development of therapies that target MYC.

Abstract Oncogenes can induce inflammatory signaling, leading to the recruitment of immune cells to tumor site. Immune cells can further influence tumor development by either restraining or facilitating tumorigenesis. Here, we investigated how distinct oncogenes shape the immune responses during tumorigenesis in vivo. We utilized several established mouse models of liver cancer in which tumorigenesis is driven by the overexpression of MYC, MET, or AKT+RAS oncogenes. We evaluated the immune cells at early vs. late stages of tumor development by immunohistochemical staining of liver tissues using lineage-specific immune cell markers. In addition, we assessed activation of the pro-inflammatory NFκB pathway by immunoblot detection of IkBα and phospho-Ser(536)-NFκB levels in liver lysates, as well as by immunohistochemical analysis of NFκB (p65) nuclear localization. Histological analysis revealed leukocytic clustering around hepatocytes in pre-neoplastic livers in MYC and AKT+RAS, but not in the MET liver tumor model. Moreover, the leukocyte profiles identified in pre-neoplastic livers and liver tumors were different in MYC vs. AKT+RAS model. Finally, we found that the NFκB pathway was inactive in MYC and AKT+RAS tumors, but hyperactivated in MET tumors. In summary, our results suggest that the type of tumor-initiating oncogenic lesion profoundly influences the identity of recruited immune cells and activation of NFκB pathway in the course of tumorigenesis. We are currently in the process of elucidating the mechanisms behind oncogene-mediated regulation of immune responses, which may reveal novel targets for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 310. doi:1538-7445.AM2012-310

The altered metabolism of tumors has been considered a target for anticancer therapy. However, the relationship between distinct tumor-initiating lesions and anomalies of tumor metabolism in vivo has not been addressed. We report that MYC-induced mouse liver tumors significantly increase both glucose and glutamine catabolism, whereas MET-induced liver tumors use glucose to produce glutamine. Increased glutamine catabolism in MYC-induced liver tumors is associated with decreased levels of glutamine synthetase (Glul) and the switch from Gls2 to Gls1 glutaminase. In contrast to liver tumors, MYC-induced lung tumors display increased expression of both Glul and Gls1 and accumulate glutamine. We also show that inhibition of Gls1 kills cells that overexpress MYC and catabolize glutamine. Our results suggest that the metabolic profiles of tumors are likely to depend on both the genotype and tissue of origin and have implications regarding the design of therapies targeting tumor metabolism.

Over-expression of Notch receptors and aberrant Notch signaling have been reported in both preinvasive ductal carcinoma in situ and invasive breast cancer. The Notch 4 receptor, in particular, is preferentially activated in human breast cancer stem cell-enriched populations, and inhibition of Notch 4 reduces tumor formation of human breast cancer cells in xenograft models (Harrison, 2010). Myc gene amplification has also been found in about 15% of breast tumors, and 22%–35% of tumors exhibit overexpression of Myc at the transcriptional level. Transgenic mice over-expressing MYC develop invasive adenocarcinomas and preferentially harbor secondary activating mutations in KRas . We examined whether Notch receptors were activated in mouse mammary tumors over-expressing MYC and activated KRAS (KRASV12) and found Notch 4 to be significantly activated in these tumors compared to normal mammary tissue. Furthermore, Notch 4 was localized to the nucleus in the tumors over-expressing MYC and KRASV12, but not in mammary glands over-expressing MYC or activated KRASV12 alone. To examine the requirement of Notch4 in mammary tumorigenesis by MYC and KRASV12, we transplanted mammary epithelial cells isolated from Notch4 knockout mice and wild-type mice that have been transduced with MYC and KRASV12 into the mammary glands of syngeneic mice. There was a significant reduction in the onset of tumorigenesis in mammary glands that had been transplanted with mammary epithelial cells lacking Notch4. These findings suggest that Notch4 is important in mammary tumorigenesis induced by cooperation of MYC and activated RAS. We are currently examining the relevance of Nocth4 activation in human breast cancers that over-express MYC and the mechanism by which Notch4 promotes tumorigenesis by Myc and activated RAS. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-01.