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Biological importance of Gab1 in HNSCC cells

Preclinical and clinical studies have evidenced that effective targeted therapy treatment designed against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, reactivation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found head and neck squamous cell carcinoma (HNSCC) was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCCs, resulting in downregulation of mTORC signaling and antitumor effects across several HNSCC mouse models, including an human papillomavirus (HPV+) patient-derived xenograft. Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2–GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. In the presence of SHP2 inhibitor, HNSCCs are highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin.Significance:This work identifies a novel role of SHP2 inhibitor by dual downregulation of PI3K and MEK pathways, through loss of GAB1 activation and disruption of GAB1 complexes in low-epiregulin HNSCC.

Cell line information and SHP099 screen data

GSK-J4 inhibits histone demethylation of EWS-FLI1 target genes

Supplemental Figure 3. Chemotherapy and venetoclax combination cell viability assays. Cell viability assays were performed for 72h treatment of cyclophosphamide and topotecan, venetoclax, or combination at indicated concentrations, with bliss synergy scores in (A) MYCN-amplified, and (B) MYCN-wild-type cell lines. (C) Plot of Bliss sum of cell lines in A-B, calculated by the summation of the bliss synergy scores across the doses presented.

Supplemental Figure 2. PDX ex vivo model demonstrated cleavage of PARP with corresponding NOXA upregulation (A) Western blot of COG-N-561 ex vivo cell line treated overnight with antibodies as indicated.

Gene symbols and names

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The sequences of the primers used for ChIP

PDF file 764K, Supp. Figures 1-10 include data describing in vitro and in vivo experiments in support of Faber at al

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Supplemental Figure 4. Body Weight of Mice for in vivo treatments. (A) Plot of percent change in body weight from pre-treatment body weight in all in-vivo treatments.

Supplemental Figure 1. NVP-CGM097 and venetoclax combination cell viability assays. Cell viability assays were performed for 16h treatment of NVP-CGM097, venetoclax, or combination at indicated concentrations, with bliss synergy scores in (A) MYCN-wild-type, p53-wild-type, (B) MYCN-amplified, p53-mutant, and (C) MYCN-wild-type, p53-mutant NB cell lines. Red indicates synergy, blue indicates antagonism. (D) Plot of Bliss sum of cell lines in A-C along with cell lines in Fig. 1B, calculated by the summation of the bliss synergy scores across the doses presented.

mp3 file (8.8 MB). In the September edition of the Cancer Discovery podcast, Executive Editor Mark Landis talks with Jeffrey A. Engelman about his paper, which suggests that quantitation of pretreatment RNA levels of the pro-apoptotic factor BIM can predict the efficacy of tyrosine kinase inhibitor therapy in oncogene-addicted cancers.

The sequences of the primers used for qPCR

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1–driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1–driven transcriptome.

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Supplemental Table 1 shows venetoclax screen data (AUC and IC50) across 791 solid tumor cell lines.

Materials & Methods

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Supplemental legend

EWS-FLI1 increases BCL-2 expression in EWFT.

Purpose:Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL.Experimental Design:We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both in vitro experiments and in vivo studies on patient-derived xenograft models.Results:We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax in vitro and leads to tumor regressions in vivo.Conclusions:Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.

Purpose:BRAFV600E mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Although selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAFV600E CRC.Experimental Design: We examined phosphoinositide 3-kinase (PI3K)/mTOR signaling in BRAFV600E CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAFV600E CRC.Results: Western blot analysis revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAFV600E GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression.Conclusions: We have established a novel GEMM to interrogate BRAFV600E CRC biology and identify more efficacious treatment strategies. Combination BRAF and PI3K/mTOR inhibitor treatment should be explored in clinical trials. Clin Cancer Res; 19(10); 2688–98. ©2013 AACR.

Sup. Table 2. Top 3 functional pathways from ingenuity pathways analysis (IPA) in DTC.

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Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M. We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M.Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo.Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo.Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658–72. ©2018 AACR.

ZEB1 binding sites found in 1975R2 cells compared to 1975 parental cells

Supplementary Figure 1 PDF file 118K, Development a GEMM for BRAFV600E CRC

Figure S4. SLC40A1 promoter and first exon are highly methylated in the MYCN overexpressing NBs.

Supplemental Figure 1. Venetoclax sensitivity and BCL-2 mRNA correlation analysis; Supplemental Figure 2. Cell viability assays of border line venetoclax-sensitive and - resistant SCLC cell lines and combination of venetoclax with chemotherapy; Supplemental Figure 3. Correlation analysis of BCL-2 family members to venetoclax sensitivity in SCLC cell lines; Supplemental Figure 4. Comparison of BCL-2 family members to venetoclax sensitivity in SCLC cell lines; Supplemental Figure 5. BCL-2 mRNA expression is increased in SCLC compared to other cancer types; Supplemental Figure 6. Cell viability of venetoclax-sensitive and -resistant SCLC cell lines; Supplemental Figure 7. Weight profiles of xenograft and PDX models treated with venetoclax; Supplemental Figure 8. The PDX models from this study express high levels of BCL-2.

Purpose:It was recently demonstrated that the EWSR1-FLI1 t(11;22)(q24;12) translocation contributes to the hypersensitivity of Ewing sarcoma to PARP inhibitors, prompting clinical evaluation of olaparib in a cohort of heavily pretreated Ewing sarcoma tumors. Unfortunately, olaparib activity was disappointing, suggesting an underappreciated resistance mechanism to PARP inhibition in patients with Ewing sarcoma. We sought to elucidate the resistance factors to PARP inhibitor therapy in Ewing sarcoma and identify a rational drug combination capable of rescuing PARP inhibitor activity.Experimental Design:We employed a pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenograft (PDX) and cell line xenograft models.Results:We found olaparib sensitivity was diminished following chemotherapy. The matched cell line pair revealed increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apoptotic resistance to olaparib. Resistance to olaparib was maintained in this chemotherapy-resistant model in vivo, whereas the addition of the BCL-2/XL inhibitor navitoclax led to tumor growth inhibition. In 2 PDXs, olaparib and navitoclax were minimally effective as monotherapy, yet induced dramatic tumor growth inhibition when dosed in combination. We found that EWS-FLI1 increases BCL-2 expression; however, inhibition of BCL-2 alone by venetoclax is insufficient to sensitize Ewing sarcoma cells to olaparib, revealing a dual necessity for BCL-2 and BCL-XL in Ewing sarcoma survival.Conclusions:These data reveal BCL-2 and BCL-XL act together to drive olaparib resistance in Ewing sarcoma and reveal a novel, rational combination therapy that may be put forward for clinical trial testing.

Figure legends for Supplementary Figures S1-S8.

Figure S1. DTCs upregulate TORC1-mediated MCL-1 translation. Figure S2. High-content cell imaging assay. Figure S3. Genetic inhibition of MCL-1 is sufficient to induce apoptosis following EGFR inhibitor treatment. Figure S4. EGFR mutant tumors (PC9) respond to EGFR inhibitor/MCL-1 inhibitor (S63845) combination therapy in vivo. Figure S5. EGFR mutant tumors (HCC827) respond to EGFR inhibitor/MCL-1 inhibitor (S63845) combination therapy in vivo. Figure S6. Upregulation of MCL-1 in EGFR mutant cell lines occurs with other insults.

MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer–related deaths. Therapy improvements for this subtype of cancer are a high priority. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1. Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. This dependence creates a marked vulnerability to targeting the system Xc-/glutathione (GSH) pathway with ferroptosis inducers. This reliance can be exploited through therapy with FDA-approved rheumatoid arthritis drugs sulfasalazine (SAS) and auranofin: in MYCN-amplified, patient-derived xenograft models, both therapies blocked growth and induced ferroptosis. SAS and auranofin activity was largely mitigated by the ferroptosis inhibitor ferrostatin-1, antioxidants like N-acetyl-L-cysteine, or by the iron scavenger deferoxamine (DFO). DFO reduced auranofin-induced ROS, further linking increased iron capture in MYCN-amplified neuroblastoma to a therapeutic vulnerability to ROS-inducing drugs. These data uncover an oncogene vulnerability to ferroptosis caused by increased iron accumulation and subsequent reliance on the system Xc-/GSH pathway.Significance:This study shows how MYCN increases intracellular iron levels and subsequent GSH pathway activity and demonstrates the antitumor activity of FDA-approved SAS and auranofin in patient-derived xenograft models of MYCN-amplified neuroblastoma.

Supplemental data

Purpose: Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor, often metastatic at presentation, for which current chemotherapeutic regimens are largely ineffective. As its pathogenesis is still unknown, we hypothesized that deregulation of signaling pathways commonly activated in cancer may contribute to MCC tumorigenesis and may provide insights into targeted therapy approaches for this malignancy.Experimental Design: We retrospectively profiled 60 primary MCC samples using a SNaPshot-based tumor genotyping assay to screen for common mutations in 13 cancer genes.Results: We identified mutations in 9 (15%) MCC primary tumors, including mutations in TP53 (3 of 60) and activating mutations in the PIK3CA gene (6 of 60). Sanger sequencing of the primary MCC tumors detected one additional PIK3CA mutation (R19K) that had not been previously described in cancer. Merkel cell polyoma virus (MCPyV) was detected in 38 (66%) MCC cases and patients with MCPyV-positive cancers showed a trend toward better survival. With one exception, the presence of MCPyV and activating mutations in PIK3CA appeared mutually exclusive. We observed that signaling through the PI3K/pAKT pathway was active in one MCPyV-positive and in all MCPyV-negative MCC cell lines, as evidenced by AKT phosphorylation. Importantly, the presence of a PIK3CA-activating mutation was associated with sensitivity to treatment with ZST474, a specific phosphoinositide 3-kinase (PI3K) inhibitor, and to NVP-BEZ235, a dual PI3K/mTOR inhibitor, targeted agents under active clinical development.Conclusions: PI3K pathway activation may drive tumorigenesis in a subset of MCC and screening these tumors for PIK3CA mutations could help identify patients who may respond to treatment with PI3K pathway inhibitors. Clin Cancer Res; 18(5); 1227–36. ©2012 AACR.

Top 10 correlated genes to BIM (BCL2L11)

Supplementary figure legends

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it.Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance.Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes.Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Supplementary Figure 2 PDF file 152K, APC tumors do not recapitulate the sessile serrated CRC pathway

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