Rita Das

AXL Mediates Resistance to PI3Kα Inhibition by Activating the EGFR/PKC/mTOR Axis in Head and Neck and Esophageal Squamous Cell Carcinomas

Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous cell carcinomas (SCCs) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance, we have observed that SCC cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)-protein kinase C (PKC), which, in turn, activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance.

An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin.

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

RAD001 Enhances the Potency of BEZ235 to Inhibit mTOR Signaling and Tumor Growth

The mammalian target of rapamycin (mTOR) is regulated by oncogenic growth factor signals and plays a pivotal role in controlling cellular metabolism, growth and survival. Everolimus (RAD001) is an allosteric mTOR inhibitor that has shown marked efficacy in certain cancers but is unable to completely inhibit mTOR activity. ATP-competitive mTOR inhibitors such as NVP-BEZ235 can block rapamycin-insensitive mTOR readouts and have entered clinical development as anti-cancer agents. Here, we show the degree to which RAD001 and BEZ235 can be synergistically combined to inhibit mTOR pathway activation, cell proliferation and tumor growth, both in vitro and in vivo. RAD001 and BEZ235 synergized in cancer lines representing different lineages and genetic backgrounds. Strong synergy is seen in neuronal, renal, breast, lung, and haematopoietic cancer cells harboring abnormalities in PTEN, VHL, LKB1, Her2, or KRAS. Critically, in the presence of RAD001, the mTOR-4EBP1 pathway and tumorigenesis can be fully inhibited using lower doses of BEZ235. This is relevant since RAD001 is relatively well tolerated in patients while the toxicity profiles of ATP-competitive mTOR inhibitors are currently unknown.

The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation

The brain microenvironment triggers HER3-dependent de novo resistance to therapies targeting PI3K or HER2 in HER2-positive and/or PIK3CA-mutant breast cancer cells. No safe haven for metastases Although targeted therapies for cancer offer great promise, they are often much less effective against brain metastases than against peripheral tumors. This is generally attributed to the drugs’ difficulty in penetrating the blood-brain barrier, but Kodack et al. now demonstrate that this is not the only reason. The authors discovered that, at least in breast cancer, the brain microenvironment itself plays a role in treatment resistance in metastatic tumors. Using mouse models and human cancer samples, the researchers found increased expression of human epidermal growth factor receptor 3 (HER3) in breast cancer–associated brain lesions and showed that it facilitates the tumors’ survival in the presence of targeted treatment and that inhibiting can help overcome resistance to therapy. Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.

Abstract 2733: LJM716: an anti-HER3 antibody that inhibits both HER2 and NRG driven tumor growth by trapping HER3 in the inactive conformation.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL HER3 (ErbB3) is a member of the ErbB family of receptor tyrosine kinases (RTK). Inappropriate HER2/ HER3 dimerization as a result of HER2 or neuregulin (NRG) over-expression in cancer results in HER3 mediated activation of PI3K signaling. Consequently, HER3 is a mediator of oncogenic transformation. Although, ligand blocking HER3 antibodies inhibit growth of neuregulin driven xenograft models they are ineffective in models of HER2 amplified cancer as HER2 mediated activation of HER3 occurs in a ligand- independent manner. LJM716 is a high affinity HER3- targeted antibody selected from a Human Combinatorial Antibody Library (HuCAL) specifically for its ability to neutralize multiple modes of HER3 activation. LJM716 is a potent inhibitor of HER3/ AKT phosphorylation and proliferation in a range of HER2 amplified and NRG expressing cell lines in vitro. LJM716 induced tumor regression in Fadu (NRG expressing, HNSCC) tumor xenografts and significant tumor growth inhibition (>80%) in a variety of xenograft models including BT474 (HER2 amplified breast). Furthermore, the combination of LJM716 with trastuzumab, cetuximab or PI3K- targeted agents was synergistic in a panel of in vitro cell lines while the in vivo combination of LJM716 with trastuzumab or erlotinib was efficacious in BT474 and L3.3 (pancreatic) tumor xenografts respectively. To further understand the mechanism by which LJM716 inhibits multiple modes of HER3 activation we solved the crystal structure of LJM716 bound to the HER3 extra-cellular domain. This data revealed that LJM716 binds to a novel conformational epitope contained within domains 2 and 4 and appears to trap HER3 in the inactive conformation. Interestingly, LJM716 does not block NRG binding to HER3 nor does it affect the binding affinity of the HER3/ NRG interaction. LJM716 therefore possesses a novel mechanism of action; it prevents the structural rearrangements required for HER3 activation induced by either HER2 or NRG. Thus LJM716 is the first HER3 antibody to display efficacy in both HER2 and ligand driven xenograft models. Based on preclinical data, combining LJM716 with either trastuzumab, cetuximab or PI3K- targeted agents may lead to greater and more sustained clinical efficacy in ErbB driven cancers. 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 2733. doi:1538-7445.AM2012-2733

Abstract 4261: Targeting HER3 and PI3K in head and neck squamous cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide, with more than 40,000 new cases developing annually in the US alone. 80-90% of HNSCC cases display elevated EGFR expression. Cetuximab an anti-EGFR monoclonal is an approved treatment of HNSCC. However, patient response to cetuximab is variable. Activation of the HER2/3 and/or the down stream PI3K pathway is a potential mechanism leading to cetuximab resistance in HNSCC. LJM716 is a fully human IgG1 ati-HER3 monoclonal antibody. BYL719 is a PI3Ka-selective small molecule inhibitor. Here, we profiled a panel of HNSCC cell lines for 1) the activation status of the EGFR/HER2/3/PI3K pathway; 2) their response to LJM716 and/or to BYL719 in combination with cetuximab. Methods: In this study, we evaluated LJM716 or BYL719 anti-tumor activity either alone, combined, or their respective combination with cetuximab in head and neck squamous cell cancer. LJM716, cetuximab and BYL719 single agent as well as combination activity of LJM716/cetuximab or LJM716/BYL719 were assessed in 33 head and neck cell lines using the CellTiter-Glo® Luminescent Cell Viability Assay. Single agent and combination anti-tumor activities of LJM716 were also tested in selected xenograft mouse models. To explore predictive markers for LJM716 and BYL719, baseline EGFR/HER2/3 and PI3K activation status was further analyzed. NRG1 level was assessed based on Affymetrix U133 plus2 genechip array. Results: Both BYL719 and LJM716 displayed anti-tumor activity in HNSCC cell lines in vitro, with BYL719 superior in most of the lines tested. There was minimum combination benefit observed with the combination of LJM716 and BYL719. Both reagents further enhanced cetuximab activity and expanded the number of cell lines sensitive to cetuximab. Anti-tumor activity was also observed with LJM716 or BYL719 in mouse xenograft models either as single agent or in combination with cetuximab. There was no correlation between baseline NRG1 or HER3 phosphorylation level with in vitro LJM716 or BYL719 efficacy. Conclusion: HNSCC is a disease highly dependent on PI3K activity. Mechanisms leading to sustained PI3K activity in HNSCC include genomic changes of PIK3CA/PTEN, activation of HER3 and additional undefined molecular events. Combining HER3/PI3K targeted therapies with cetuximab may overcome some of the resistance to cetuximab therapy in HNSCC. Future work is underway to identify biomarkers that would predict response to these combination regimens. Citation Format: Qing Sheng, HuiQin Wang, Rita Das, Yan Chen, Jinsheng Liang, Alex Cao, Carl Uli Bialucha, Seth Ettenberg, Alan Huang. Targeting HER3 and PI3K in head and neck squamous 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 4261. doi:10.1158/1538-7445.AM2013-4261

Abstract 1061: Modulation of death pathways by TRAILR2 agonist antibody and NF-κB pathway by NIK inhibitor in HPV-positive head and neck squamous cell carcinomas

Head and neck squamous cell carcinomas (HNSCC) induced by human papilloma virus (HPV) have increased recently in the US, and exhibit a different prognosis and response to therapies from HPV(-) cancers. Analysis of HNSCC TCGA datasets provide evidence for distinct alterations in expression of components of the NF-κB and death pathways in HNSCC with different HPV status. Previously, we have found that birinipant, a novel SMAC mimetic that inhibits inhibitor of apoptosis proteins (IAPs), sensitizes a subset of HPV(-) HNSCC cell lines to death agonists like TNF-α and TRAIL. In this study, we have observed that birinipant also sensitizes most HPV(+) cell lines to TRAIL and TNF-α in vitro. The IC50 of birinipant was under 50nM for HPV(+) UPCI-SCC-90 and UM-SCC-47 cell lines, when combined with TNF-α or TRAIL. To explore the therapeutic potential of enhancing TRAILR mediated death signaling in HPV(+) HNSCC cells, we investigated the effects of an agonistic polyclonal TRAILR2 antibody. Treatment of cells with TRAILR2 antibody alone shows little or no inhibitory effect on UPCI-SCC-90 and UM-SCC-47 cells in vitro. However, a combination of birinipant and TRAILR2 antibody, or triple combination of birinipant, TRAIL, and TRAILR2 antibody shows additive or synergistic effects to inhibit cell proliferation and induce cell death in a dose dependent manner. In addition, our preliminary data suggested that the non-canonical NF-κB pathway is predominately activated in HPV(+) HNSCC cells, and that NF-κB inducing kinase (NIK) is a key component of this pathway. When we tested the NIK inhibitor 4H-isoquinoline-1,3-dione in UPCI-SCC-90 cells, we found it reduced cell proliferation in a dose-dependent manner (IC50 =1.5 μM). Taken together, these results indicate that TNF-α, TRAIL, and TRAILR2 agonist antibody sensitized birinipant anti-tumor activity, and triple combination exhibited synergistic effects in HPV(+) HNSCC cell lines. NIK inhibitor alone inhibits cell proliferation of HPV(+) cells, supporting the hypothesis of aberrant activation of alternative pathway in HPV(+) HNSCC cells. (Supported by NIDCD intramural projects ZIA-DC-000016, 73, 74). Citation Format: Yi An, Lillian Sun, Adeeb Derakhshan, Sophie Carlson, Rita Das, Zhong Chen, Carter Van Waes. Modulation of death pathways by TRAILR2 agonist antibody and NF-κB pathway by NIK inhibitor in HPV-positive head and neck squamous cell carcinomas [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 1061. doi:10.1158/1538-7445.AM2017-1061

Abstract 5008: The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation

Brain metastases represent a devastating progression of luminal breast cancer. While targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade PI3K inhibition despite efficient drug delivery. In comparison to extracranial disease, there is increased HER3 expression and phosphorylation in the brain lesions. HER3 blockade overcomes the resistance of both HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, leading to striking tumor growth delay and significant improvement of mouse survival. Collectively, these data provide a mechanistic basis underlying therapeutic resistance in the brain microenvironment and identify rapidly translatable treatment strategiesfor HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases. Citation Format: Gino B. Ferraro, David P. Kodack, Vasileios Askoxylakis, Qing Sheng, Mark Badeaux, Shom Goel, Xiaolong Qi, Ram Shankaraiah, Alexander Z. Cao, Rakesh R. Ramjiawan, Divya Bezwada, Bhushankumar Patel, Youngchul Song, Carlotta Costa, Kamila Naxerova, Christina Wong, Jonas Kloepper, Rita Das, Angela Tam, Jantima Tanboon, Dan G. Duda, Ryan C. Miller, Marni B. Siegel, Carey K. Anders, Melinda Sanders, Valeria M. Estrada, Robert Schlegel, Carlos L. Arteaga, Elena Brachtel, Alan Huang, Dai Fukumura, Jeffrey A. Engelman, Rakesh K. Jain. The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation [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 5008. doi:10.1158/1538-7445.AM2017-5008

Abstract 2904: The alternative NF-kB2/RELB pathway is activated by LTB/LTB receptor and NIK to promote cell migration and metastasis-related gene expression in HNSCC

Head and neck squamous cell carcinomas (HNSCC) are highly inflammatory and preferentially migrate and metastasize to lymph nodes. In this study, we find that LTβ/LTβR/NIK signaling mediates alternative RELB/NF-kB2 activation, which promotes activation of important cancer related genes and migration. We find that LTβ and LTβR are over expressed in subsets of HNSCC tissues and cell lines, and LTβ activates the alternative NF-κB pathway, enhancing nuclear translocation of RELB and NF-kB2/p52. Knockdown of LTβR decreased its target kinase NIK, and downstream NF-kB subunits RELB and NF-kB2/p52 protein expression. Knockdown of NIK protein decreased RELB and p52 protein expression, while LTβ treatment stabilized NIK, RELB and NF-kB2/p52 expression. Consistent with this, knockdown of LTBR and NIK functionally decreased NF-kB reporter gene activity, while treatment of LTB partially restored the NF-kB reporter activity. Notably, knockdown of NIK and RELB by siRNA inhibited cell migration in HNSCC. Since NIK is known as central regulator of the activation of the non-canonical pathway, we tested the effects of a NIK inhibitor 1,3[2H,4H]-Isoquinolinedione on NF-kB function and cell migration. NIK inhibitor decreased NIK protein in the cytoplasm, downstream nuclear expression of RELB and NF-kB2/p52 proteins by Western blot and RELB localization by immunofluorescence staining. We have found evidence for LTβ induction of migration/metastasis and cell death genes MET, BIRC3,and SERPINE1, and NIK knockdown inhibited cell migration and the inducible expression of MET, BIRC3, SERPINE 1 by LTB. Our data reveal the mechanisms how LTβ and NIK activation mediated alternative NF-kB pathway and contribute to migration and metastasis of HNSCC. Supported by NIDCD intramural Project ZIA-DC-000016, 73 and 74. Citation Format: Rita Das, Tsu-Fan Cheng, Jamie Coupar, Anthony Saleh, Paul E. Clavijo, Xinping Yang, Zhong Chen, Carter VanWaes. The alternative NF-kB2/RELB pathway is activated by LTB/LTB receptor and NIK to promote cell migration and metastasis-related gene expression in HNSCC. [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 2904.

Abstract LB-237: Targeting HER3 and IGF1R in NRG1 high lung squamous cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA HER3 is a member of the ErbB family of receptor tyrosine kinases. Aberrant activation of HER3 as a result of HER2 amplification or neuregulin 1 (NRG1) over-expression has been demonstrated to mediate constitutive activation of downstream oncogenic signals. LJM716 is a fully human IgG1 anti-HER3 monoclonal antibody. It locks HER3 in an inactive conformation and prevents HER3 dimerization with other ErbB family members. This unique mode of action enables LJM716 to block both ligand dependent and ligand independent HER3 activation. In search of new indications that may benefit from anti-HER3 therapies, we noted that squamous cell carcinomas (SCC) tend to have relatively higher NRG1 expression as compared to their adenocarcinoma counterparts. Highest NRG1 expression was observed in the lung SCC cell lines in cell line encyclopedia (CLE). A subset of the lung SCC cell lines with high NRG1 expression are moderately sensitive to LJM716, with HER3 activation detected in all of the sensitive lines. Surprisingly, phosphorylation of IGF1R rather than other HER family members tracked closely to HER3 phosphorylation in the lung SCC lines. Consistent with this finding, co-treatment with either IGF1R inhibitor, OSI-906, or anti-IGF1R antibody, Figitumumab, with LJM716 further enhanced LJM716 activity in the three NRG1-high lung SCC lines in vitro. The enhanced anti-tumor activity of LJM716 and Figitumumab is also observed in mouse xenografts generated from these lung SCC lines. Furthermore, pharmacodynamic analysis revealed increased inhibition of PI3K/mTOR signaling with the combined treatment of LJM716 and Figitumumab. These data suggest that NRG1- driven HER3 activation is a common feature in a subset of lung squamous cell carcinoma. Combination of anti-HER3 and anti-IGF1R is effective at suppressing PI3K/mTOR signaling and inhibit growth in these tumors. Citation Format: Qing Sheng, Maria Pinzon-Ortiz, Rita Das, Alan Huang, Xianhui Rong, Z. Alexander Cao. Targeting HER3 and IGF1R in NRG1 high lung squamous cell carcinoma. [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 LB-237. doi:10.1158/1538-7445.AM2014-LB-237