Meenal Chalukya

Expression of p16 and Retinoblastoma Determines Response to CDK4/6 Inhibition in Ovarian Cancer

Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients. Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052). Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

Therapeutic Potential of the Poly(ADP-ribose) Polymerase Inhibitor Rucaparib for the Treatment of Sporadic Human Ovarian Cancer

Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer. Mol Cancer Ther; 12(6); 1002–15. ©2013 AACR.

Frequent EVI1 translocations in myeloid blast crisis CML that evolves through tyrosine kinase inhibitors

Clinical variables associated with ecotropic viral integration site 1 (EVI1) translocations were evaluated in 42 consecutive chronic myeloid leukemia (CML) patients in myeloid blast crisis (MBC). Translocations were confirmed with fluorescence in situ hybridization, and Western blot analysis demonstrated EVI1 expression. Translocations of EVI1 were present in 3 of 24 (12%) patients whose disease evolved MBC before tyrosine kinase inhibitor (TKI) exposure, and 7 of 18 (39%) patients who had received one or more TKIs. Univariate analysis showed that prior TKI therapy was the only clinical variable that was significantly associated with EVI1 translocation (P = 0.047). TKI-resistant BCR-ABL1 mutations were present in 71% of MBC patients with EVI1 translocations at the time of disease progression. These observations suggest that EVI1 overexpression collaborates with BCR-ABL1 in the evolution of TKI-resistant MBC. Inhibition of c-ABL kinase-mediated DNA double-strand repair by TKIs may predispose to EVI1 translocation in this setting.

Abstract 25: PD 0332991, a selective CDK 4/6 inhibitor, preferentially inhibits growth of ovarian cancer cells with high Rb and low p16 (CDKN2A) expression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Cell cycle dysregulation is a common molecular finding in ovarian cancer and the cyclin-D kinases (CDK) represent an attractive target in this pathway. PD 0332991 (Pfizer Inc.) is an orally active potent and highly selective inhibitor of CDK 4 and CDK6 kinases with the ability to block pRb phosphorylation at serine 780 and 795 in the low nanomolar range. To identify predictors of response to PD 0332991, we determined the in vitro sensitivity to PD 0332991 in a large panel of molecularly characterized ovarian cancer cell lines, and then used baseline expression profiles and Western blot analyses to identify biomarkers associated with response to PD 0332991. These biomarkers were then validated in a large cohort of 324 ovarian cancer patients by immunohistochemistry. Methods: 40 human ovarian cancer cell lines were treated with PD 0332991 using two-fold dilutions over 12 concentrations. Dose response curves were generated using a cell count assay to calculate the IC50 across the panel. These data were then analyzed against baseline gene expression data to identify genes associated with sensitivity to PD 0332991. Western blot analysis was performed to validate these markers and to study the effects of PD 0332991 on pRb phosphorylation. Cell cycle analysis was performed using flow-cytometry. Combination studies were performed to analyze the interaction between PD 0332991 and paclitaxel or carboplatin chemotherapy. Tissue microarrays were constructed from 324 ovarian cancer patients and IHC was performed for p16 and Rb expression. Long-term clinical follow up was available for all patients. Results: Concentration-dependent anti-proliferative effects of PD 0332991were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC50s (IC50 range: 0.02 to >20 μmol/L). Cell lines with high Rb expression and low p16 expression by gene expression array and Western blot analyses were most sensitive to inhibition by PD 0332991. Cell lines treated with PD 0332991 showed clear G0/G1 arrest and a decrease in S-phase fraction in sensitive but not in resistant cell lines. In addition, Western blot showed that pRb phosphorylation is blocked in a concentration and time dependent manner. IHC for Rb and p16 allows the identification of ovarian cancer patients most likely to benefit from CDK4/6 inhibition. Outcome analyses are ongoing. Conclusion: PD 0332991 shows promising biologic activity in ovarian cancer cells. Assessment of Rb and p16 expression may help select patients who benefit from therapeutic strategies targeting CDK4/6 in ovarian cancer. 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 25.

Abstract 1795: Potent anti-tumor activity of talazoparib (BMN673) in combination with radiation for squamous cell carcinoma of the head and neck

Background: Squamous cell carcinoma of the head and neck (SCCHN) is a radiation-sensitive disease. Standard frontline treatment of locally advanced SCCHN entails concurrent chemotherapy with radiation. Treatment of relapsed or metastatic SCCHN often utilizes re-irradiation to the primary tumor for local control. However, there is no standard systemic treatment for relapsed or metastatic SCCHN in combination with radiation. Many SCCHN tumors display alterations in expression of DNA repair genes. To this end, we evaluated the effect of talazoparib (BMN-673), a potent poly-ADP ribose polymerase (PARP) inhibitor, alone or in combination with radiation, in vitro and in vivo. Methods: The 50% inhibitory concentration (IC50) of talazoparib was determined in 30 SCCHN cell lines in vitro. Using clonogenic assays, the effects of 0, 10 or 100nM talazoparib, alone or in combination with a single fraction of 4Gray radiation were determined in four cell lines (two sensitive and two resistant to talazoparib). Effects on DNA damage of talazoparib, alone or in combination with radiation were also determined by flow cytometric analysis of Gamma-H2A.X. Finally, the FaDu head and neck cells were implanted onto female J/nude. After tumors were established, mice were treated with talazoparib for 5 days and a total of 20 Gray radiation, alone or in combination, and effects on body weight and tumor size were determined. Results: Among 30 SCCHN cell lines treated with talazoparib alone, 18 displayed sensitivity with 50% inhibitory concentration (IC50) of 1μM or less. In clonogenic assays, addition of radiation to BMN673 in two sensitive (UMSCC-6 and UMSCC-38) and two resistant (UMSCC-5 and UMSCC-12) cell lines resulted in synergistic cytotoxicity at doses ranging from 0-100nM BMN673. Increased gamma-H2A.X was seen when cell lines were treated with talazoparib, radiation, or the combination, compared to controls. Combining radiation with talazoparib resulted in a transient decrease in body weight in J/nude mice, but resulted in statistically significant and prolonged decrease in tumor volume. These anti-tumor effects were sustained out to 60 days post inoculation. Conclusions: The PARP inhibitor talazoparib with radiation synergistically inhibited tumor growth in vitro and in vivo in SCCHN cell lines. Further evaluation of this combination is underway. Citation Format: Deborah JL Wong, David D. Shin, Josephine Ratikan, Meenal Chalukya, Kanthinh Manivong, Leonard Post, Dorthe Schaue, Richard S. Finn, Yuqiao (Jerry) Shen, William McBride, Dennis J. Slamon. Potent anti-tumor activity of talazoparib (BMN673) in combination with radiation for squamous cell carcinoma of the head and neck. [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 1795. doi:10.1158/1538-7445.AM2015-1795

Abstract 743: Potent anti-tumor activity of the MEK1/2 inhibitor MEK162 in human non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: MAPK pathway dysregulation is frequently seen in cancer. For NSCLC, this may be due to driver mutations or gene amplification in EGFR, KRAS, or BRAF, though tumors without such mutations frequently display pathway hyperactivity. Aberrant EGFR signaling in SCCHN may lead to hyperactivity of the MAPK or PI3K pathways. MEK162 (ARRY-438162) is a potent, tight binding, uncompetitive MEK inhibitor currently in clinical development for treatment of solid tumors. MEK162 has an IC50 of 10nM against purified MEK1/2. We evaluated whether MEK162 would inhibit NSCLC and SCCHN cell lines and whether specific molecular alterations predicted anti-tumor activity. We also evaluated the combination of MEK162 in combination with BYL719, a potent PI3K alpha inhibitor. METHODS: The 50% inhibitory concentration (IC50) of MEK162, alone or in combination with BYL719, was determined in 40 human NSCLC and 30 SCCHN cell lines in vitro. Effects on MAPK and PI3K/AKT pathway signaling were studied by western blot analysis. Effects of MEK162 alone or in combination with BYL719 on cell cycle progression were also determined by flow cytometry. RESULTS: Using previously published cut-offs for sensitivity in cell lines, among NSCLC cell lines, 20/40 (50%) were sensitive to MEK162 (IC50 ≤ 500nM), 9/40 were intermediately sensitive (IC50 500nM-1µM), and 11/40 were resistant (IC50 ≥ 1µm). Using the same cut-offs, 80% (24/30) of SCCHN cell lines were sensitive, 2/30 were intermediately sensitive, and 4/30 were resistant to MEK162. MEK162 exposure resulted in increased cells in G0/G1 and decreased cells in S-phase. MEK162 decreased phosphorylated ERK (p-ERK) in all cell lines treated and resulted in an increase in phosphorylated AKT (p-AKT), presumably as a compensatory result from MAPK pathway inhibition. Combined treatment with MEK162 and BYL719 resulted in synergistic growth inhibition for all cell lines and decreased levels of both p-ERK and p-AKT levels at 24 hours. CONCLUSION: MEK162 potently inhibited growth of human NSCLC and SCCHN cell lines in most cell lines tested and led to synergistic inhibition when combined with BYL719. The compensatory increase in pAKT seen with MEK162 was abrogated with dual treatment with MEK162 and BYL719, demonstrating that synergy may be mediated by simultaneous blockade of MAPK and PI3K/AKT pathways. These data provide a preclinical rationale for evaluating MEK162, alone or in combination with BYL719, in metastatic NSCLC and SCCHN. Supported by 1K23CA149079, P50 CA090440, V Foundation for Cancer Research, Jonsson Comprehensive Cancer Center, Wolfen Family Lung Cancer Research Program, Stiles Program in Oncology, National Lung Cancer Partnership and One Ball Matt Memorial Golf Tournament. Citation Format: Deborah JL Wong, Edward B. Garon, Danielle D. Silveira, Naeimeh Kamranpour, Sharon Pitts, Meenal Chalukya, Habib Hamidi, Steven Dubinett, Ronald Linnartz, Richard S. Finn, Dennis J. Slamon. Potent anti-tumor activity of the MEK1/2 inhibitor MEK162 in human non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) cell lines. [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 743. doi:10.1158/1538-7445.AM2014-743

Abstract 1595: The poly(ADP-ribose) polymerase inhibitor BMN 673 has single agent activity and augments cytotoxicity of radiation in human head and neck tumor cell linein vitro: a novel strategy for radiosensitization in head and neck cancer.

Background and purpose: Current treatment of head and neck cancer generally involves a combination of surgery, radiation and chemotherapy. Clinical outcomes for many patients remain suboptimal, and novel therapeutic strategies are needed. Targeted inhibition of poly(ADP-Ribose) polymerase (PARP), a key DNA repair enzyme, has been actively investigated in cancer medicine. BMN 673 is a potent orally active inhibitor of PARP, currently in phase I clinical trials in solid and hematologic malignancies. We hypothesized BMN 673 might have single agent activity in a subset of head and neck cancers and could significantly augment the effects of radiation in this disease. To test this strategy, we performed preclinical studies to evaluate single agent activity of BMN 673 in a large panel of human head and neck cell lines. We also aimed to define interactions between BMN 673 and radiation in these models. Materials and methods: Human head and neck cell lines (N=32) were treated with single-agent BMN 673 for 5 days, and in vitro growth inhibition assays were performed. Further, four cell lines were studied in colony formation assays by seeding and treating them with BMN 673 at 1, 10, or 100 nM (or vehicle) for 24 hours, followed by mock or 4 Gy irradiation (300kV, 10mA). Cells were incubated for 14 or 20 days and stained with crystal violet for colony enumeration. Each experiment was performed in triplicates. Survival fraction was calculated: (number of colonies for treated cells/number of cells plated)/(number of colonies for corresponding control/number of cells plated). Results: A gradation of sensitivity was observed with BMN 673 monotherapy: IC 50 ranges from 0.1 to 10 μM. Radiation alone reduced colony forming ability of the cells by 26% in UMSCC-5 and by approximately 65% in other three cell lines (UMSCC-6, 12, and 38). These cells exhibited enhanced cytotoxicity with addition of BMN 673 to radiation in a dose-dependent manner. Addition of 100nM of BMN 673 resulted in 80% reduction in the number of surviving colonies in the relatively radio-resistant UMSCC-5 and in 88-100% reduction in other cell lines. All changes were statistically significant. Conclusions: In vitro assays demonstrate human head and neck cancer cells exhibit relative growth sensitivity to BMN 673 monotherapy. In addition, radiation-induced cytotoxicity is augmented by the addition of BMN 673 to radiation in a dose-dependent manner. Notably, one relatively radio-resistant line was rendered radio-sensitive with the addition of BMN 673 to radiation. These data support a role of PARP inhibition as monotherapy and/or in combination with radiotherapy, representing a potential novel translational therapeutic strategy in head and neck cancers that warrant clinical testing. Citation Format: David D. Shin, Josephine Ratikan, Kanthinh Manivong, Meenal Chalukya, Leonard Post, Yuqiao (Jerry) Shen, William McBride, Dorthe Schaue, Dennis J. Slamon, Richard S. Finn. The poly(ADP-ribose) polymerase inhibitor BMN 673 has single agent activity and augments cytotoxicity of radiation in human head and neck tumor cell line in vitro: a novel strategy for radiosensitization in head and neck cancer. [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 1595. doi:10.1158/1538-7445.AM2013-1595