C Williams

Abstract CT053: High rates of personalized molecular matching are achievable in a precision oncology navigation trial: the I-PREDICT study

Precision medicine has evolved as an individualized approach for treating cancer patients and has become standard in an ever-increasing number of clinical settings. It is predicated upon matching targeted-/immuno-therapy to genomic alterations detected in patients9 tumors. However, widespread feasibility/adoption has been limited by: 1) high rates of insufficient tumor DNA (reaching 25%); 2) panels limited to few genes that are unable to detect multiple classes of genomic alterations; 3) testing patients late in the disease course; and 4) low molecular matching rates, which may be in part due to limited access to trials and the unpredictable nature of genomic alterations detected in each individual. We evaluated the feasibility of investigating molecular profile-related evidence for determining individualized cancer therapy (I-PREDICT) in patients with lethal tumors (NCT02534675). This navigation trial was performed under the auspices of 2 precision medicine programs (UCSD and Avera Cancer Institute) and an IRB-approved protocol. Treatment-naive and previously treated patients with ECOG Citation Format: Jason K. Sicklick, Brian Leyland-Jones, Shumei Kato, Casey Williams, Pradip De, Gregory Heestand, Steven Plaxe, Benjamin Solomon, Vincent Miller, Adam Benson, Jennifer Webster, Jeffrey Ross, Michael Scur, Robert Porter, Shelby Jepperson, Paul Fanta, Razelle Kurzrock. High rates of personalized molecular matching are achievable in a precision oncology navigation trial: the I-PREDICT study [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 CT053. doi:10.1158/1538-7445.AM2017-CT053

Abstract P5-03-14: MLN0128 regulates survival signaling by AKT and its downstream effectors in HER2+ breast cancer model

Evading apoptosis is considered to be a hallmark of cancers including breast cancer, since mutations in apoptotic regulators invariably accompany tumorigenesis. Chemotherapeutic agents induce apoptosis, and hence disruption of apoptosis during tumor progression may promote drug resistance. AKT is an apoptotic regulator that is activated in HER2+ breast tumor cells and promotes anti-HER2 therapy resistance in vitro . Nevertheless, how mTORC1/C2-AKT signaling disables apoptosis and its contribution to clinical drug resistance are not clear yet. Using HER2 amplified breast cancer cells [BT474 ( HER2+/ Trastuzumab-sensitive), BT474HerR ( HER2+/ Trastuzumab-resistant), HCC1954 and MDA-MB453 (both are HER2 +/ PIK3CA kinase domain mutated)], we show that mTORC1/C2 inhibitor; MLN0128 abrogates AKT (Ser473), Survivin and controls its downstream effectors of apoptotic signaling molecules (e.g. cleaved Caspase 3/9, cleaved PARP, MCL and BIM). MLN0128 also induces annexinV positive cells and regulates cellular proliferation (ON-TOP 3D colony formation and real-time proliferation assay). Additionally, increased cleaved Caspase 3 and decreased MCL1 expression were also observed following MLN0128 treatment in HER2+ xenograft model along with tumor growth inhibition. Our studies provide strong experimental evidence that high apoptotic signaling –specifically reduced MCL1 and increased cleaved-CASPASE3 expression expedite the response of targeted therapy that directly inhibits mTORC1/C2-AKT signaling. Citation Format: De PK, Carlson JH, Sun Y, Lin X, Williams C, Dey N, Leyland-Jones BR. MLN0128 regulates survival signaling by AKT and its downstream effectors in HER2+ breast cancer model. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-14.

Abstract P3-14-08: Preclinical efficacy of targeting c-MET by ARQ197 in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines

Background: Triple-negative breast cancer (TNBC), accounts for 15% of all invasive breast cancers (BCs) and has the poorest survival outcome of all BC subtypes. Due to its heterogeneity, TNBC lacks validated therapeutic targets compared with other BC subtypes (Sohn et al., 2014; Foulkes et al., 2010). Therefore, improved approaches to treatment of these cancers are unmet needed. Several molecular targets including: epidermal growth factor receptor (EGFR), poly ADP ribose polymerase (PARP), and hepatocyte growth factor receptor (c-MET) are under clinical investigation for the treatment of this disease (De et al., 2014; Cleator et al., 2007). The MET oncogene encodes a membrane-bound tyrosine kinase implicated in the formation and/or progression of several cancer types including TNBC, and several studies have shown c-Met overexpression to be an independent predictor of poor outcome in BC (Ho-Yen et al., 2014), c-MET may play a critical role in the development of the most aggressive BCs and may be a rational therapeutic target (Graveel et al., 2009). Currently inhibitors targeting c-MET (including ARQ197) are undergoing clinical trials in a variety of cancers including TNBC (Gaule et al., 2014; ClinicalTrials.gov). Recently, PARP inhibitors in combination with chemotherapy, has shown promising results in TNBC in clinical and preclinical studies (Tutt et al., 2010; De et al., 2014). We argue that, blocking the PARP-mediated nuclear machinery for repairing DNA-damage in presence of cytotoxic DNA damaging agents in conjunction with co-targeting c-Met pathway dependent downstream effectors may have a robust anti-tumor activity in TNBC cell lines. Methodology: BT-20 (PIK3CA mutated, H1047R), HCC70 (PTEN null), HCC1937 (PTEN null), MDA-MB-231 (KRAS/BRAF mutated), MDA-MB-468 (PTEN null) and SUM149PT (BRCA1 mutated) cells were used for this study. Growth inhibition, survival/proliferation, colony formation and apoptosis were examined using MTT assay, 2D proliferative /growth assay, 3D-ON-TOP assays, and annexinV staining respectively. Results: 1) For all TNBC cell lines, the IC50 of single agent ARQ197 was from 0.5 µM to 1.5 µM (following 96 hours treatment) 2) ARQ197 as a single agent or in combination with ABT888 or in triple combination dose dependently decreased cell growth/proliferation 3) annexin V positive cells were increased following treatment with single agent ARQ197 or in combination with ABT888 or in triple combination 4) 70-99% anti-proliferative activities were observed on 3D-ON TOP colony formation assay with ARQ197 alone or in combination in all tested cell lines. Conclusion: Our preclinical in vitro drug sensitivity data suggest that administration of c-MET inhibitor may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC models. Mechanism studies are ongoing, the results of which will be presented in the meeting. Citation Format: Sun Y, Carlson JH, Lin X, De PK, Williams C, Hausman S, Dey N, Leyland-Jones BR. Preclinical efficacy of targeting c-MET by ARQ197 in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-08.

Abstract P4-08-04: Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine

Background: Treatment of BC is conventionally based on the presence/absence of ER/PR or HER2 status of the primary tumor. We have enriched this approach by including major genetic and proteomic changes in tumors of individual patients in order to develop a better treatment-rationale based on an alteration driven signaling algorithm. Methods: Genomic and proteomic data from 75 BC patients seen in our center were retrospectively analyzed. Patients were re-biopsied after consultation and samples were characterized (IHC for ER, PR, and HER2; FFPE samples for genomic [Foundation Medicine] and proteomic analyses [Theranostics]). In vivo studies were conducted using xenograft models. Results: Although alterations of PIK3CA, PIK3R1, AKT, PTEN, MDM2, MDM4, TSC1, mTOR and RICTOR are most frequently observed in our patients, there is a distinct pattern of alteration(s) of the PI3K pathway genes in different subtypes of BC. A total of 76 genes were altered in 48 ER+BC patients. In 79% of ER+BC patients the above mentioned PI3K pathway genes were altered. Analyzing the set of alterations of genes in individual patients, we observed that within these 48 patients 25% exhibited alterations in more than one node of the pathway; the most common combination (alterations) being the amplification/mutation of PIK3CA with the amplification of MDM2/4 genes. The percentage of patients belonging to HER2+ & TNBC exhibiting similar alterations in the PI3K pathway genes were significantly lower (∼40%). Our previous in vivo studies demonstrated that GDC-0980 and BEZ235 enhanced the antitumor activity of ABT888 plus carboplatin in TNBC or trastuzumab in HER2+ BC respectively and blocked the growth of established xenograft tumors by 80% to 90% with a concomitant decrease in tumor Ki67, pS6RP and CD31. Mechanistically the action of the PI3K-mTOR pathway targeted drug(s) was tested using cell line based models of BC subtypes pertaining to their respective genomic alterations. A combination of a pan-PI3K pathway inhibitor, GDC-0941 or isoform-specific inhibitors along with AI, trastuzumab, or HRD inhibitors (PARP) blocked proliferative signals and enhanced apoptosis (cleaved caspase3) in ER+/PIK3CA mutated, HER2+/PIK3CA mutated or PTEN-null TNBC cells respectively as demonstrated by WB, flow cytometry, cell proliferation, viability and cytotoxicity assays. A recent study demonstrated that exposure to chemotherapy induced a phenotypic shift or cell state transition towards a transient CD44Hi/CD24Hi chemotherapy-tolerant state, leading to the activation of downstream non-receptor tyrosine kinase signaling towards an emerging adaptive resistance (Goldman et al., Nature Comm. 2015). Hence drug combination(s) are being tested for their effect on CD44/CD24 expression levels, results of which will be presented in the meeting. Conclusion: Plotting the genetic alterations from the patient on the signaling landscape will be useful in cracking the code leading to improved treatment options. Patient specific in-depth plotting of genetic alterations of the PI3K-mTOR pathway and the relevance of these alterations in the context of (1) mechanisms of PI3K-mTOR pathway targeted drugs and (2) cell signaling are critical in determining choice of drugs in BC subtypes. Citation Format: Carlson JH, Krie A, Williams C, Sun Y, Lin X, Williams K, Klein J, Friedman L, De P, Dey N, Leyland-Jones B. Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-04.

Abstract 4222: Targeting PI3K and mTOR with BEZ235, a dual inhibitor, is efficacious in trastuzumab refractory and HER2+/PIK3CA-mutated breast cancer models

Background: PI3K pathway activation is a common event in HER2+ breast cancer (BC) either through inactivation of the tumor suppressor gene, PTEN, or activation of PIK3CA. This PIK3CA mutation leads to an oncogenic activity of the PI3K pathway and contributes to therapeutic resistance to trastuzumab (T). The PI3K pathway inhibitor is therefore an attractive drug for HER2+ BC, especially in T-resistant and PIK3CA mutated conditions. Methodology: Here we have studied the in vitro and in vivo effects of BEZ235 in HER2+/T-sensitive (BT474), HER2+/T-resistant (BT474HerR) and HER2+/PIK3CA (HCC1954) mutated models. We assessed in vitro anti-proliferative and activation status of the PI3K-AKT-mTOR signaling pathway following BEZ235 treatment in HER2+ BC cell lines. We next evaluated the impact of BEZ235 on tumor growth and angiogenesis using xenograft models. Results: 1) BEZ235 inhibited downstream activation of the PI3K signaling pathway effectors, p-AKT (Ser473, The308), p-P70S6K, p-S6RP and p-4EBP1, 2) the anti-proliferative activity of BEZ235 was observed by 3D-ON-TOP clonogenic assay, 3) consistent with anti-proliferative effects of BEZ235, the proportion of cells in the G1 phase of the cell cycle increased in all three cell lines with a concomitant decrease in the S phase of their treatment with BEZ235, 4) the initiation of apoptotic activity (annexin V) of BEZ235 was significantly superior to that of RAD001, an allosteric inhibitor of mTOR, and 5) in the HER2+/T-sensitive, HER2+/T-resistant and HER2+/PIK3CA mutated BC xenograft models, BEZ235 inhibited PI3K signaling and had potent anti-tumor activity. Along with its anti-tumor effect, BEZ235 effectively decreased tumor angiogenesis (tumor micro-vessel density via CD31 staining). These inhibitions were more pronounced when BEZ235 was combined with T. Conclusions: BEZ235 inhibits the PI3K-AKT-mTOR signaling pathway and results in anti-proliferative and anti-tumor activity in HER2+ breast cancer cells with both wild type and mutated PIK3CA. Citation Format: Casey Williams, Yuliang Sun, Jennifer Carlson, Brian Leyland-Jones, Nandini Dey, Pradip De. Targeting PI3K and mTOR with BEZ235, a dual inhibitor, is efficacious in trastuzumab refractory and HER2+/ PIK3CA- mutated breast cancer models. [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 4222. doi:10.1158/1538-7445.AM2014-4222