Joan Cao

An integrated genomic approach to identify predictive biomarkers of response to the aurora kinase inhibitor PF-03814735.

PF-03814735 is a novel, reversible inhibitor of Aurora kinases A and B that finished a phase I clinical trial for the treatment of advanced solid tumors. To find predictive biomarkers of drug sensitivity, we screened a diverse panel of 87 cancer cell lines for growth inhibition upon PF-03814735 treatment. Small cell lung cancer (SCLC) and, to a lesser extent, colon cancer lines were very sensitive to PF-03814735. The status of the Myc gene family and retinoblastoma pathway members significantly correlated with the efficacy of PF-03814735. Whereas RB1 inactivation, intact CDKN2A/p16, and normal CCND1/Cyclin D1 status are hallmarks of SCLC, activation or amplification of any of the three Myc genes (MYC, MYCL1, and MYCN) clearly differentiated cell line sensitivity within the SCLC panel. By contrast, we found that expression of Aurora A and B were weak predictors of response. We observed a decrease in histone H3 phosphorylation and polyploidization of sensitive lines, consistent with the phenotype of Aurora B inhibition. In vivo experiments with two SCLC xenograft models confirmed the sensitivity of Myc gene-driven models to PF-03814735 and a possible schedule dependence of MYC/c-Myc–driven tumors. Altogether our results suggest that SCLC and other malignancies driven by the Myc family genes may be suitable indications for treatment by Aurora B kinase inhibitors. Mol Cancer Ther; 11(3); 710–9. ©2012 AACR.

Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.

Molecular Predictors of Sensitivity to the Insulin-like Growth Factor 1 Receptor Inhibitor Figitumumab (CP-751,871)

Figitumumab (CP-751,871), a potent and fully human monoclonal anti–insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors. Mol Cancer Ther; 12(12); 2929–39. ©2013 AACR.

Combined gemcitabine and CHK1 inhibitor treatment induces apoptosis resistance in cancer stem cell-like cells enriched with tumor spheroids from a non-small cell lung cancer cell line

Evaluating the effects of novel drugs on appropriate tumor models has become crucial for developing more effective therapies that target highly tumorigenic and drug-resistant cancer stem cell (CSC) populations. In this study, we demonstrate that a subset of cancer cells with CSC properties may be enriched into tumor spheroids under stem cell conditions from a non-small cell lung cancer cell line. Treating these CSC-like cells with gemcitabine alone and a combination of gemcitabine and the novel CHK1 inhibitor PF-00477736 revealed that PF-00477736 enhances the anti-proliferative effect of gemcitabine against both the parental and the CSC-like cell populations. However, the CSC-like cells exhibited resistance to gemcitabine-induced apoptosis. Collectively, the spheroid-forming CSC-like cells may serve as a model system for understanding the mechanism underlying the drug resistance of CSCs and for guiding the development of better therapies that can inhibit tumor growth and eradicate CSCs.

Abstract 764: Rational combination of PF-06463922 (next-generation ALK inhibitor) with PI3K pathway inhibitors overcomes ALKi resistance in EML4-ALK+ NSCLC models

Crizotinib (PF-02341066) is a small molecule tyrosine kinase inhibitor of ALK, ROS1 and c-MET that is approved in over 70 countries for the treatment of ALK fusion positive non-small cell lung cancer (ALK+ NSCLC). Crizotinib achieved robust objective response rates of approximately 60% in ALK+ NSCLC and significantly improved progression free survival compared to chemotherapy. The emergence of secondary mutations within the ALK kinase domain or the activation of compensatory signaling pathways in crizotinib and other ALKi refractory tumors prompted searches for next generation of ALKi active against resistance mutations as single agents or in combination with other treatments. Such effort led to our recent discovery of PF-06463922, an ALK/ROS1 inhibitor with greatly improved ALK potency, brain penetration, and broad spectrum activity against all known clinical ALKi-resistant mutations. PF-06463922 is being tested in a Phase I clinical trial in both ALK+ and ROS1 fusion positive NSCLC in treatment naive or ALKi relapsed patients. In our current preclinical study, we explored rational combination strategies to further improve the efficacy of PF-06463922 in ALKi resistant cells or tumors. Our results show that compared to PF-06463922 alone, the combination of this compound with PI3K pathway inhibitors, such as PF-05212384 (PI3K/mTOR), GDC0941 (pan-PI3K) or GDC0032 (beta-sparing) leads to more robust anti-proliferative activity in vitro and greater duration of efficacy in vivo in the ALKi resistant models. These PI3K pathway inhibitors also partially overcome EGF or HGF ligand-induced resistance to PF-06463922. Interestingly, in addition to AKT signaling, both compounds inhibit ERK signaling as well, which may be essential for their enhancement of PF-06463922 cell activity or tumor efficacy in combination settings. Studies are ongoing to identify optimal partners for PF-06463922 combination using isoform selective PI3Ki, AKTi and mTORi. We are also exploring the breadth of efficacy of this combination in overcoming resistance to crizotinib, PF-06463922 or other ALKi. The findings provide important evidence that will help define the clinical development path for PF-06463922. This research effort may ultimately lead to more effective approaches to treat ALKi refractory patients in the clinic. Citation Format: Ping Wei, Ming Qiu, Nathan Lee, Joan Cao, Hui Wang, Konstantinos Tsaparikos, Conglin Fan, Timothy Sargis, Justine Lam, Maruja E. Lira, Goldie Lui, James Hardwick, Valeria Fantin, Paul Rejto, Tod Smeal. Rational combination of PF-06463922 (next-generation ALK inhibitor) with PI3K pathway inhibitors overcomes ALKi resistance in EML4-ALK+ NSCLC models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 764. doi:10.1158/1538-7445.AM2015-764

Abstract 4464: Elucidation of crizotinib resistance in NCI-H3122 and strategies to circumvent bypass resistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The Anaplastic Lymphoma Kinase (ALK) fusion proteins such as EML4-ALK act as oncogenic drivers in approximately 5-7% of non-small cell lung carcinomas (NSCLC). The rationale for targeting this kinase as a means of treatment has been validated by the clinical efficacy and subsequent approval of the ALK inhibitor, crizotinib (Xalkori ®). The overall response rate of 61% suggests potential intrinsic resistance to crizotinib. In addition, like many tyrosine kinase inhibitors, acquired resistance inevitably leads to treatment refractory cancers. To better understand resistance mechanisms (both intrinsic and acquired), and circumvent resistance to crizotinib, we developed in vitro resistance models through prolonged crizotinib exposure. We isolated resistance clones and characterization identified different resistance mechanisms, including pathway bypass as well as an ALK kinase mutation (G1269A), similar to those that have been reported from refractory patient samples. The majority of the resistance clones escaped crizotinib suppression via pathway bypass. In several resistance clones, simultaneous activation of EGFR and IGF1R receptors limited the efficacy of double combination strategies and required triple combinations. In addition, phospho-proteomic analysis revealed simultaneous activation of different receptor tyrosine kinases, suggesting that combination treatment with inhibitors of downstream mediators such as PI3K and MEK may be effective strategies. Our resistance models demonstrate the heterogeneity of cancers and reveal the multiplicity of escape mechanisms. Our results provide support for the evaluation of combination therapy with downstream mediator inhibitors as a way to restore as well as enhance sensitivity to crizotinib. Citation Format: Nathan V. Lee, Joan Cao, Timothy Scheffelin, Lars Engstrom, Stephen Huang, Maruja Lira, Scott Garza, Jing Yuan, Blake Enyart, James Christensen, Julie Kan. Elucidation of crizotinib resistance in NCI-H3122 and strategies to circumvent bypass resistance. [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 4464. doi:10.1158/1538-7445.AM2013-4464

Abstract 2355: Palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in squamous cell lung cancer

Lung cancer remains one of the leading causes of cancer-related mortality. Squamous cell lung cancer (SqCLC) is the second most common subtype of non-small cell lung cancer (NSCLC) and is responsible for ~100,000 deaths in the US and EU. Most SqCLC patients receive chemotherapy as 1st line treatments and have a high un-met medical need for new therapies. Therapeutic approaches that enhance the efficacy of chemotherapy would therefore improve clinical outcomes for this patient population. CDK inhibitors comprise a class of drugs that target the dysregulated cell cycle in malignant cells. Treatment of tumor cells with the CDK4/6 inhibitor palbociclib inhibits tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Based on promising clinical trial results, palbociclib in combination with letrozole was granted accelerated approval by the US FDA for the treatment of postmenopausal women with ER-positive, HER2-negative advanced breast cancer. Like hormone receptor positive breast cancer patients, the vast majority of SqCLC patients harbor wild type RB and thus may also benefit from palbociclib treatment. Previously, we reported robust cytotoxicity and antitumor effects of palbociclib plus taxanes, including nanoparticle albumin-bound paclitaxel (Nab-PTX) or docetaxel (DTX), in several preclinical models of SqCLC. In the present study, we extended our efficacy studies of this combination to additional RB+ SqCLC models with diverse molecular genetic backgrounds. In search of mechanisms of action underlying the observed combinatorial effects, we identified several novel mechanisms, including cell cycle checkpoint abrogation as well as reduction of hypoxia-inducible factor 1 alpha (HIF-1α). Decrease in HIF-1α protein led to strong modulation of downstream genes involved in angiogenesis, resulting in reduced blood vessel size in tumor vasculature. Our results suggest that palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in SqCLC. Citation Format: Joan Cao, Zhou Zhu, Hui Wang, Tim Nichols, Edward Rosfjord, Christine Hopf, Erik Upeslacis, Paul Rejto, Scott Weinrich, Todd Vanarsdale, James Hardwick, Ping Wei. Palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in squamous cell lung cancer [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 2355. doi:10.1158/1538-7445.AM2017-2355

Abstract A43: RB pathway disruption by the CDK4/6 inhibitor palbociclib enhances responses to chemotherapy in squamous cell lung cancer

Lung cancer remains one of the leading causes of cancer-related mortality. Squamous cell lung cancer (SqCLC) is the second most common subtype of non-small cell lung cancer (NSCLC). Despite recent development of effective targeted therapeutic agents for lung adenocarcinoma, patients with SqCLC often receive conventional cytotoxic chemotherapy as this cancer subtype lacks genomic alterations that can be targeted by personalized medicine. Hence, novel approaches that enhance the efficacy of chemotherapy will benefit treatment outcomes in this patient population. CDK inhibitors comprise a class of drugs that targets the dysregulated cell cycle in malignant cells. Treatment of tumor cells with the CDK4/6 inhibitor palbociclib inhibits tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Based on promising clinical trial data, palbociclib in combination with letrozole was granted accelerated approval by the US FDA for the treatment of postmenopausal women with ER-positive, HER2-negative advanced breast cancer. In this preclinical study, we explored the effect of palbociclib on several chemotherapies (taxanes, platins, and antimetabolites) in preclinical models of SqCLC. Because the activity of chemotherapy generally requires cell cycle progression, careful combination/sequencing of this class of drugs with CDK inhibitors may be important to achieve synergy as well as avoid potential antagonism. To obtain optimal activity of palbociclib and chemotherapy combinations, we investigated several combination/sequencing regimens (concurrent, chemotherapy followed by palbociclib or the reverse sequence) in several SqCLC cell lines. We did not encounter antagonism of chemotherapy-mediated cytotoxicity by palbociclib in any of the tested regimens. Rather, we observed robust combinatorial anti-cancer cell activity in all settings. Combination of palbociclib with chemotherapy was associated with reduction of RB phosphorylation and FOXM1 protein levels, and the induction of p21. Our studies demonstrated that, while palbociclib partially antagonized chemotherapy-induced apoptosis, it significantly synergized with chemotherapy to induce cell cycle arrest as well as a senescence-like phenotype. Cells pretreated with palbociclib plus cisplatin or palbociclib plus docetaxel displayed less cell growth upon drug removal compared to those treated with monotherapies. Finally, palbociclib treatment that followed docetaxel, nab-paclitaxel or cisplatin treatment significantly enhanced the antitumor activity of the chemotherapies in several cell line-derived or patient-derived xenograft models. Our results suggest that treatment with optimal palbociclib and chemotherapy combination/sequencing could lead to better clinical outcomes for SqCLC patients. Citation Format: Ping Wei, Joan Cao, Goldie Lui, Hui Wang, Konstantinos Tsaparikos, David Shields, Kim Arndt, Paul Rejto, Todd VanArsdale, James Hardwick. RB pathway disruption by the CDK4/6 inhibitor palbociclib enhances responses to chemotherapy in squamous cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A43.

Abstract 2817: Preclinical studies of the PI3K/mTOR dual inhibitors in endometrial cancer cell lines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The PI3K pathway plays a pivotal role in many cellular functions that include regulation of cell proliferation, survival, growth, angiogenesis, and motility. Dysregulation of the PI3K/AKT pathway is a common occurrence in cancer and could be a result of mutations in PIK3CA and PTEN, as well as loss of heterozygosity of PTEN. In endometrial cancer patients, the most frequent aberration is the activation of the PI3K/mAKT pathway with; (1) 80% loss of PTEN function and 30% PIK3CA mutations in type I endometrial cancer and (2) 20% PIK3CA mutation and 46% PIK3CA amplification in type II endometrial cancer. In this study, we investigated the functional consequences of the dual PI3K/mTOR inhibitors PF-04691502 and PF-05212384 in endometrial cancer cell lines for antiproliferative effects, pathway signaling inhibition and tumor growth inhibition. Both compounds had antiproliferative activity in vitro that translated to tumor growth inhibition in vivo. Moreover, in the MFE-280 xenograft model that harbors a PIK3CA mutation (P1047R), the dual PI3K/mTOR inhibitors showed tumor regression. These studies support the use of the dual PI3K/mTOR inhibitors for endometrial cancer. 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 2817. doi:1538-7445.AM2012-2817

Abstract LB-302: Activity of PF-04691502, A dual PI3K/mTOR inhibitor in breast cancer cell lines and models discriminates between ER, PR and HER2 positive and negative segments

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The PI3K pathway plays a role in key cellular functions that include regulation of cell growth, proliferation, survival, angiogenesis, and motility. PI3K/AKT pathway aberrations are common in cancer comprising PTEN loss of function through mutations, deletion, and methylation events and gain of function at the PIK3CA locus including mutations and amplification events resulting in deregulation of this pathway. Thus, pharmacological intervention of this pathway should impact cellular functions central to survival of cancer cells. PIK3CA mutations/amplification and PTEN loss of heterozygosity have been reported to occur in approximately 35-40% and 20% of breast cancer, respectively and suggest this patient population could benefit from treatment using a small molecule inhibitor that targets the PI3K pathway. PF-04691502 which is a potent inhibitor of all PI3K isoforms and mTOR (TORC1 and TORC2) presently in Phase 1 trials was evaluated for its growth inhibitory or cytoreductive activity over a panel of 30 breast cancer cell lines. PF-04691502 had robust <300nM (IC50%) antiproliferative activity across the panel of breast cell lines. We observed a trend whereby cell lines that were hormone receptor (ER and/or PR) and HER2 negative (triple negative) were least sensitive and cell lines that were HER2(+) and/or hormone receptor positive (ER and/or PR) were more sensitive to PF-04691502. These studies were extended in vivo with PF-04691502 as a single agent and in combination in a HER2, ER and PR positive xenograft model with robust tumor growth inhibition in combination with docetaxel or with the pan-HER inhibitor, PF-00299804. In a xenograft that was triple negative, administration of single agent PF-4691502 at the MTD resulted in 60% tumor growth inhibition. In conclusion, our findings suggest that the ER/PR and/or HER2 positive breast cancer segment may particularly benefit from PI3K/mTOR inhibitor based treatment regimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-302.