Tuhina Mazumdar

Kinase-Impaired BRAF Mutations in Lung Cancer Confer Sensitivity to Dasatinib

Induction of tumor cell senescence may explain the response of a patient with BRAF kinase–impaired lung cancer to the multikinase inhibitor dasatinib. A Lucky Break with BRAF The prognosis for those with metastatic non–small cell lung cancer (NSCLC) is bleak—the median survival time is measured in months. Therapeutic benefits have been achieved with targeted drugs in subpopulations of NSCLC patients with specific mutations, but the genetic changes responsible for this disease are undefined in most cases. Understanding why certain tumors respond to a given treatment might help determine useful therapeutic targets. Sen et al. now describe a striking case—the mutation responsible for the strong response of one patient with metastatic NSCLC to treatment with the tyrosine kinase inhibitor dasatinib. In a previous clinical trial of dasatinib treatment for metastatic NSCLC that lasted for 12 weeks, only a single patient responded to treatment; his tumor shrank and continued to shrink after treatment ended. Four years later, he appears free of active cancer. The researchers analyzed this patient’s tumor tissue and did not detect mutations that had been associated with NSCLC in other patients, but did find a mutation in the serine-threonine kinase BRAF that markedly impaired its kinase activity. (In contrast, another well-characterized oncogenic mutation in BRAF is kinase-activating.) Sen et al. found that in NSCLC cell lines with other kinase-inactivating BRAF mutations, dasatinib induced largely irreversible senescence—cell cycle arrest. Overexpression of kinase-active BRAF, however, increased dasatinib resistance in these cells, indicating that the inactive BRAF kinase was required for their dasatinib sensitivity. Furthermore, treatment of dasatinib-resistant cancer cells that express wild-type BRAF with a BRAF inhibitor increased their sensitivity to dasatinib. Exactly how dasatinib induces senescence in NSCLC cells with kinase-impaired BRAF is not yet clear, but the finding opens new possibilities for treatment. Cancers in which BRAF is impaired may respond well to dasatinib; more broadly, dasatinib in combination with BRAF inhibitors may be useful for treating tumors that express wild-type BRAF. During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non–small cell lung cancer (NSCLC), one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase-inactivating BRAF mutation (Y472CBRAF); no inactivating BRAF mutations were found in the nonresponding tumors taken from other patients. Cells transfected with Y472CBRAF exhibited CRAF, MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase), and ERK (extracellular signal–regulated kinase) activation—characteristics identical to signaling changes that occur with previously known kinase-inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells’ dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by Y472CBRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization, resulting in ERK activation in NSCLC cells with kinase-inactivating BRAF mutations. The sensitivity of NSCLC with kinase-impaired BRAF to dasatinib suggested synthetic lethality of BRAF and an unknown dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells’ dasatinib sensitivity. Thus, the patient’s BRAF mutation was likely responsible for his tumor’s marked response to dasatinib, suggesting that tumors bearing kinase-impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF.

A Comprehensive Evaluation of Biomarkers Predictive of Response to PI3K Inhibitors and of Resistance Mechanisms in Head and Neck Squamous Cell Carcinoma

The PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), but pathway inhibition has variable efficacy. Identification of predictive biomarkers and mechanisms of resistance would allow selection of patients most likely to respond and novel therapeutic combinations. The purpose of this study was to extend recent discoveries regarding the PI3K/AKT/mTOR pathway in HNSCC by more broadly examining potential biomarkers of response, by examining pathway inhibitors with a diverse range of targets, and by defining mechanisms of resistance and potential combination therapies. We used reverse-phase protein arrays (RPPA) to simultaneously evaluate expression of 195 proteins; SNP array to estimate gene copy number; and mass array to identify mutations. We examined altered signaling at baseline and after pathway inhibition. Likewise, we examined the activation of the PI3K/AKT/mTOR pathway in HNSCC tumors by RPPA. Cell lines with PIK3CA mutations were sensitive to pathway inhibitors, whereas amplification status did not predict sensitivity. While we identified a set of individual candidate biomarkers of response to pathway inhibitors, proteomic pathway scores did not correlate with amplification or mutation and did not predict response. Several receptor tyrosine kinases, including EGFR and ERK, were activated following PI3K inhibition in resistant cells; dual pathway inhibition of PI3K and EGFR or MEK demonstrated synergy. Combined MEK and PI3K inhibition was markedly synergistic in HRAS-mutant cell lines. Our findings indicate that clinical trials of single-agent PI3K/AKT/mTOR pathway inhibitors in selected populations and of PI3K/EGFR or PI3K/MEK inhibitor combinations are warranted; we plan to conduct such trials. Mol Cancer Ther; 13(11); 2738–50. ©2014 AACR.

Tumor grafts derived from patients with head and neck squamous carcinoma authentically maintain the molecular and histologic characteristics of human cancers

BackgroundThe patient-derived xenograft (PDX) model is likely to reflect human tumor biology more accurately than cultured cell lines because human tumors are implanted directly into animals; maintained in an in vivo, three-dimensional environment; and never cultured on plastic. PDX models of head and neck squamous cell carcinoma (HNSCC) have been developed previously but were not well characterized at the molecular level. HNSCC is a deadly and disfiguring disease for which better systemic therapy is desperately needed. The development of new therapies and the understanding of HNSCC biology both depend upon clinically relevant animal models. We developed and characterized the patient-derived xenograft (PDX) model because it is likely to recapitulate human tumor biology.MethodsWe transplanted 30 primary tumors directly into mice. The histology and stromal components were analyzed by immunohistochemistry. Gene expression analysis was conducted on patient tumors and on PDXs and cell lines derived from one PDX and from independent, human tumors.ResultsFive of 30 (17%) transplanted tumors could be serially passaged. Engraftment was more frequent among HNSCC with poor differentiation and nodal disease. The tumors maintained the histologic characteristics of the parent tumor, although human stromal components were lost upon engraftment. The degree of difference in gene expression between the PDX and its parent tumor varied widely but was stable up to the tenth generation in one PDX. For genes whose expression differed between parent tumors and cell lines in culture, the PDX expression pattern was very similar to that of the parent tumor. There were also significant expression differences between the human tumors that subsequently grew in mice and those that did not, suggesting that this model enriches for cancers with distinct biological features. The PDX model was used successfully to test targeted drugs in vivo.ConclusionThe PDX model for HNSCC is feasible, recapitulates the histology of the original tumor, and generates stable gene expression patterns. Gene expression patterns and histology suggested that the PDX more closely recapitulated the parental tumor than did cells in culture. Thus, the PDX is a robust model in which to evaluate tumor biology and novel therapeutics.

Dasatinib induces DNA damage and activates DNA repair pathways leading to senescence in non-small cell lung cancer cell lines with kinase-inactivating BRAF mutations

Improved therapies are greatly needed for non-small cell lung cancer (NSCLC) that does not harbor targetable kinase mutations or translocations. We previously demonstrated that NSCLC cells that harbor kinase-inactivating BRAF mutations (KIBRAF) undergo senescence when treated with the multitargeted kinase inhibitor dasatinib. Similarly, treatment with dasatinib resulted in a profound and durable response in a patient with KIBRAF NSCLC. However, no canonical pathways explain dasatinib-induced senescence in KIBRAF NSCLC. To investigate the underlying mechanism, we used 2 approaches: gene expression and reverse phase protein arrays. Both approaches showed that DNA repair pathways were differentially modulated between KIBRAF NSCLC cells and those with wild-type (WT) BRAF. Consistent with these findings, dasatinib induced DNA damage and activated DNA repair pathways leading to senescence only in the KIBRAF cells. Moreover, dasatinib-induced senescence was dependent on Chk1 and p21, proteins known to mediate DNA damage-induced senescence. Dasatinib also led to a marked decrease in TAZ but not YAP protein levels. Overexpression of TAZ inhibited dasatinib-induced senescence. To investigate other vulnerabilities in KIBRAF NSCLC cells, we compared the sensitivity of these cells with that of WTBRAF NSCLC cells to 79 drugs and identified a pattern of sensitivity to EGFR and MEK inhibitors in the KIBRAF cells. Clinically approved EGFR and MEK inhibitors, which are better tolerated than dasatinib, could be used to treat KIBRAF NSCLC. Our novel finding that dasatinib induced DNA damage and subsequently activated DNA repair pathways leading to senescence in KIBRAF NSCLC cells represents a unique vulnerability with potential clinical applications.

Drug-induced RAF dimerization is independent of RAS mutation status and does not lead to universal MEK dependence for cell survival in head and neck cancers

Treatments for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) have limited efficacy. One potential therapeutic target for HNSCC is the RAS/RAF/MEK/ERK cascade, which is one of the major signaling pathways for HNSCC cell survival. In HNSCC, RAS can be activated either by HRAS mutation or by upstream signaling. The ABL inhibitor nilotinib acts as a weak RAF inhibitor that induces RAF dimerization and subsequent activation of MEK/ERK in other cancer cell lines with activated RAS, leading to an unexpected dependence on MEK/ERK for cell survival. We hypothesized that nilotinib and the MEK inhibitor MEK162 would be synergistic in HNSCC cell lines owing to the frequent activation of RAS. We treated HNSCC cell lines with nilotinib and performed immunoblotting and cell-viability experiments. We used an orthotopic mouse model to assess synergistic effects in vivo. Nilotinib induced significant BRAF-CRAF heterodimerization and ERK activation irrespective of RAS mutation status. In cell-viability assays, nilotinib synergized with MEK162. MEK162 alone induced G1 arrest that was minimally enhanced by nilotinib. In the mouse model, treatment with MEK162 alone or combined with nilotinib led to tumor growth inhibition. In HNSCC, nilotinib-induced RAF dimerization is independent of RAS mutation status, but this dimerization does not lead to MEK dependence for cell survival in all HNSCC cell lines. MEK inhibition alone leads to decreased proliferation both in vitro and in vivo. Although nilotinib has some synergistic effects with MEK162, other agents may be more effective against HNSCC when combined with MEK162.

Genomic characterization of human papillomavirus-positive and -negative human squamous cell cancer cell lines

Human cancer cell lines are the most frequently used preclinical models in the study of cancer biology and the development of therapeutics. Although anatomically diverse, human papillomavirus (HPV)-driven cancers have a common etiology and similar mutations that overlap with but are distinct from those found in HPV-negative cancers. Building on prior studies that have characterized subsets of head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESC) cell lines separately, we performed genomic, viral gene expression, and viral integration analyses on 74 cell lines that include all readily-available HPV-positive (9 HNSCC, 8 CESC) and CESC (8 HPV-positive, 2 HPV-negative) cell lines and 55 HPV-negative HNSCC cell lines. We used over 700 human tumors for comparison. Mutation patterns in the cell lines were similar to those of human tumors. We confirmed HPV viral protein and mRNA expression in the HPV-positive cell lines. We found HPV types in three CESC cell lines that are distinct from those previously reported. We found that cell lines and tumors had similar patterns of viral gene expression; there were few sites of recurrent HPV integration. As seen in tumors, HPV integration did appear to alter host gene expression in cell lines. The HPV-positive cell lines had higher levels of p16 and lower levels of Rb protein expression than did the HPV-negative lines. Although the number of HPV-positive cell lines is limited, our results suggest that these cell lines represent suitable models for studying HNSCC and CESC, both of which are common and lethal.

Abstract 4095: Paradoxical activation of the RAF-MEK-ERK pathway in response to nilotinib induces synthetic lethality with MEK inhibition in head and neck cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cancers of the head and neck are difficult to treat because both the therapy and the tumor impact essential functions such as speech and swallowing and they can significantly alter facial appearance. The RAS/RAF/MEK/ERK cascade is one of the major signaling pathways that leads to head and neck squamous cell carcinoma (HNSCC) cell survival. There are three RAS genes (HRAS, KRAS, and NRAS) and together, they are mutated in about 30% of human cancers. RAS can also be activated by upstream signaling. Paradoxically RAF inhibition induces RAF-dimerization and causes subsequent activation of MEK/ERK pathway in presence of activated RAS in melanoma. Similarly the ABL inhibitor nilotinib acts as weak inhibitor of RAF kinase and also induces RAF-dimerization and subsequent activation of MEK/ERK in the presence of activated RAS in leukemia and pancreatic cancer cells. Nilotinib synergizes with MEK inhibitition to kill nilotinib-resistant leukemia cells (Abl T351I). We hypothesized that nilotinib and the MEK inhibitor MEK162 would be synergistic in HNSCC due to the frequent activation of RAS via upstream growth factor receptors. RAS mutations are relatively uncommon in HNSCC. We screened 60 validated HNSCC cell lines through sequenome analysis and we found three HNSCC cell lines with activating HRAS mutations (G12D and Q61L) [∼5%]. We found that nilotinib induces significant RAF dimerization and subsequent activation of MEK-ERK pathway irrespective of RAS mutational status. We next investigated how paradoxical MEK/ERK pathway activation affected the growth of HNSCC cells. In this context we treated a panel of HNSCC cell lines harboring WT and mutated HRAS with nilotinib and MEK162. Nilotinib alone had nearly no effect on cell viability and MEK162 had a moderate effect on some HNSCC lines. The combination of nilotinib and MEK162 synergized to inhibit the growth of HNSCC cells independent of RAS mutational status. The drug combination led to an accumulation of cells in G1 and a significant reduction in cell proliferation as measured with BrDU incorporation and colony formation. HNSCC cells with mutant HRAS undergo apoptosis in presence of both the drugs. In contrast none of these drugs induce senescence in HNSCC cell lines. Here we show that in HNSCC, nilotinib as single agent drives the paradoxical activation of RAF-MEK-ERK pathway in RAS mutation-independent manner and we uncovered a synthetic lethal pathway that can be used treat HNSCC. Animal studies are planned. This work was supported by University of Texas SPORE in Head and Neck Cancer P50 CA097007. Citation Format: Banibrata Sen, Tuhina Mazumdar, Shaohua Peng, Katherine S. Hale, Bonnie S. Glisson, Faye M. Johnson. Paradoxical activation of the RAF-MEK-ERK pathway in response to nilotinib induces synthetic lethality with MEK inhibition in head and neck cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4095. doi:10.1158/1538-7445.AM2013-4095

Abstract 2992: Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC)

Background: Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. However, clinical trials of PI3K/mTOR pathway inhibitors had limited success even in those tumors with pathway alterations, including PIK3CA mutations. To target genomic alterations in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and molecular characteristics in order to identify biomarkers of response. Methods: We systematically analyzed the association between drug sensitivity and genomic alterations in 59 HNSCC lines. Results: NOTCH1mut lines are significantly sensitive to PI3K/mTOR pathway inhibitors: GSK2126458 (13/16), BYL719 (6/16), PQR309 (13/16), BKM120 (14/16), BEZ235 (12/16), BAY806942 (14/16) and GDC0980 (13/16 lines). In contrast to PIK3CAmut cell lines, all 7 NOTCH1mut lines tested underwent apoptosis (14.3 fld; P Conclusion: In contrast to PIK3CAmut cells, NOTCH1mut HNSCC cells underwent apoptosis after PI3K/mTOR pathway inhibition in vitro and decreased tumor size in vivo. The ectopic activation of NOTCH1 rescued NOTCH1mut HNSCC cells from PI3K/mTOR inhibitor-mediated apoptosis. The underlying mechanism may involve differential effects on tumor metabolism and ROS production. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our future work may uncover previously unknown crosstalk between the PI3K/mTOR and NOTCH pathways in SCCs. Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Tuhina Mazumdar, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2017-2992

Abstract 393: NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma

Recently published whole exome sequencing studies in head and neck squamous cell carcinoma (HNSCC) tumors revealed that few had therapeutically targetable alterations using current strategies. This finding defines translational gap between genomics and HNSCC treatment. One potential targetable alteration is PIK3CA mutations. However, clinical trials testing PI3K/mTOR pathway inhibitors have had limited success and these inhibitors only lead to cell cycle arrest in PIK3CA mutant HNSCC cell lines. Thus, there is a critical need to identify therapeutic vulnerabilities for common mutation groups, including tumor suppressors, in HNSCC. One of these molecular subgroups is NOTCH1 which is the second most frequently mutated gene in HNSCC, with a 10-15% prevalence of inactivating mutations. Although there are several studies underscoring the importance of NOTCH1 as a tumor suppressor in HNSCC, none has identified a therapy that targets NOTCH1 mutant (mut) HNSCC. Our objective was to identify predictive biomarkers of sensitivity to PI3K/mTOR inhibitors by integrating drug and multiple-omics data. Cell viability with six PI3K/mTOR inhibitors in 68 HNSCC lines was measured by the CellTiter Glo assay. The peak plasma concentration of each drug was used as the cut-off to determine sensitivity. We observed a striking correlation between NOTCH1mut and sensitivity to PI3K/mTOR pathway inhibitors. When fisher9s exact test was performed, NOTCH1mut lines were more sensitive to GSK2126458 (P NOTCH1mut lines underwent more apoptosis after GSK2126458 treatment compared to NOTCH1wt lines (PCI15B- 48.1 fold; P Our data suggests that loss of active NOTCH1 signaling confers sensitivity to PI3K/mTOR inhibition. If the combination of NOTCH1 and PI3K/mTOR inhibition leads to apoptosis, this combination could be translated into the clinic. Citation Format: Faye M. Johnson, Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Pan Tong, Tuhina Mazumdar, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick. NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393.

Abstract 4757: Identification of biomarkers that predict response of head and neck squamous cell carcinoma to mitotic inhibitors

Objectives: It is urgent to explore novel biomarkers and therapeutic targets for that are relevant to head and neck squamous cell carcinoma (HNSCC), which is the 6th most common cancer worldwide. Based on a prior drug screen, we identified 3 mitotic inhibitors (AZD7762, AZD1775, volasertib) as effective therapies for HNSCC. Our objective with this study is to identify mechanisms of response and potential biomarkers of response and Methods: Cell viability assays were performed by the CellTiter-Glo Luminescent method in a panel of 68 fingerprinted HNSCC cell lines using the 3 drugs at concentrations of 0.018 to 9.613 μM. Cell cycle, apoptosis and altered pathway protein expression after cells treated by the polo-like kinase 1 (PLK1) inhibitor volasertib were investigated by FACS, TUNEL and western blots respectively. An orthotopic mouse model of HNSCC was used to confirm the antitumor effects of PLK1 inhibition in vivo. To determine the mechanisms of drug sensitivity, we analyzed the correlation between gene expression, protein expression, gene mutation and drug sensitivity using modified two-sample t-tests were performed. The beta-uniform mixture (BUM) model was used to control false discovery rate (FDR). For correlations between drug sensitivity and gene mutations, we performed Fisher9s exact test. Results: Using the IC80 values with the peak plasma concentration of each drug as the cut-off to determine sensitivity, 34, 44 and 20 HNSCC cell lines were sensitive AZD1775 (Wee inhibitor), AZD7762 (CHK1/2 inhibitor) and volasertib (PLK1 inhibitor) respectively. HNSCC harboring AJUBA mutations were more sensitive to these 3 inhibitors and those with RAS mutations more resistant. PLK1 inhibition led to G2/M arrest, but only sensitive cell lines underwent substantial apoptosis following PLK1 inhibition. Decreases of the levels of phosphorylated TCTP were observed following treatment with volasertib confirming PLK1 inhibition. There was a significant decrease of tumor volumes and prolongation of survival in the mice bearing orthotopic HNSCC tumors treated with volasertib in vivo. Conclusions: PLK1 inhibition was an effective therapy in vitro and in vivo models of HNSCC. We identified the AJUBA and RAS mutations as potential candidate biomarkers of response to these mitotic inhibitors in HNSCC. This study identified the therapeutic potential of PLK1 as a novel therapeutic target for HNSCC. Citation Format: Ming Zhang, Shaohua Peng, Tuhina Mazumdar, Vaishnavi Sambandam, Li Shen, Pan Tong, Lerong Li, Lauren Byers, Curtis Pickering, Mitchell Frederick, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Identification of biomarkers that predict response of head and neck squamous cell carcinoma to mitotic inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4757.