Jason P.W. Carey

AXL Inhibition Suppresses the DNA Damage Response and Sensitizes Cells to PARP Inhibition in Multiple Cancers

Epithelial to mesenchymal transition (EMT) is associated with a wide range of changes in cancer cells, including stemness, chemo- and radio-resistance, and metastasis. The mechanistic role of upstream mediators of EMT has not yet been well characterized. Recently, we showed that non–small cell lung cancers (NSCLC) that have undergone EMT overexpress AXL, a receptor tyrosine kinase. AXL is also overexpressed in a subset of triple-negative breast cancers (TNBC) and head and neck squamous cell carcinomas (HNSCC), and its overexpression has been associated with more aggressive tumor behavior and linked to resistance to chemotherapy, radiotherapy, and targeted therapy. Because the DNA repair pathway is also altered in patient tumor specimens overexpressing AXL, it is hypothesized that modulation of AXL in cells that have undergone EMT will sensitize them to agents targeting the DNA repair pathway. Downregulation or inhibition of AXL directly reversed the EMT phenotype, led to decreased expression of DNA repair genes, and diminished efficiency of homologous recombination (HR) and RAD51 foci formation. As a result, AXL inhibition caused a state of HR deficiency in the cells, making them sensitive to inhibition of the DNA repair protein, PARP1. AXL inhibition synergized with PARP inhibition, leading to apoptotic cell death. AXL expression also associated positively with markers of DNA repair across TNBC, HNSCC, and NSCLC patient cohorts. Implications: The novel role for AXL in DNA repair, linking it to EMT, suggests that AXL can be an effective therapeutic target in combination with targeted therapy such as PARP inhibitors in several different malignancies. Mol Cancer Res; 15(1); 45–58. ©2016 AACR.

PKCiota promotes ovarian tumor progression through deregulation of cyclin e

The high frequency of relapse of epithelial ovarian tumors treated with standard chemotherapy has highlighted the necessity to identify targeted therapies that can improve patient outcomes. The dynamic relationship between cyclin E and PKCiota frequent overexpression in high-grade ovarian tumors poses a novel pathway for therapeutic investigation. We hypothesized that a phosphoinositide 3-kinase (PI3K)-dependent signaling pathway activating PKCiota perpetuates cyclin E deregulation during ovarian tumorigenesis. We observed a positive correlation between PKCiota and cyclin E in a panel of 19 ovarian cancer cell lines. Modulation of cyclin E had no effect on PKCiota knockdown/overexpression; however, PKCiota differentially regulated cyclin E expression. In the serous ovarian cancer cells (IGROV and OVCAR-3), shPKCiota decreased proliferation, caused a G1 arrest and significantly prolonged overall survival in xenograft mouse models. In vitro, shPKCiota decreased the ability of IGROV cells to grow under anchorage-independent conditions and form aberrant acini, which was dependent on Ad-cyclin E or Ad-LMW-E expression. Reverse-phase protein array analysis of PKCiota wild-type, catalytic active, dominant-negative protein isoforms strengthened the association between phospho-PKCiota levels and PI3K pathway activation. Inhibitors of PI3K coordinately decreased phospho-PKCiota and cyclin E protein levels. In conclusion, we have identified a PI3K/PKCiota/cyclin E signaling pathway as a therapeutic target during ovarian tumorigenesis.

Cyclin E overexpression as a biomarker for combination treatment strategies in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a virulent form of breast cancer, and novel treatment strategies are urgently needed. Immunohistochemical analysis of tumors from women with a clinical diagnosis of IBC (n = 147) and those with non-IBC breast cancer (n = 2510) revealed that, whereas in non-IBC cases cytoplasmic cyclin E was highly correlated with poor prognosis (P < 0.001), in IBC cases both nuclear and cytoplasmic cyclin E were indicative of poor prognosis. These results underscored the utility of the cyclin E/CDK2 complex as a novel target for treatment. Because IBC cell lines were highly sensitive to the CDK2 inhibitors dinaciclib and meriolin 5, we developed a high-throughput survival assay (HTSA) to design novel sequential combination strategies based on the presence of cyclin E and CDK2. Using a 14-cell-line panel, we found that dinaciclib potentiated the activity of DNA-damaging chemotherapies treated in a sequence of dinaciclib followed by chemotherapy, whereas this was not true for paclitaxel. We also identified a signature of DNA repair–related genes that are downregulated by dinaciclib, suggesting that global DNA repair is inhibited and that prolonged DNA damage leads to apoptosis. Taken together, our findings argue that CDK2-targeted combinations may be viable strategies in IBC worthy of future clinical investigation.

Synthetic lethality of PARP inhibitors in combination with MYC blockade is independent of BRCA status in triple negative breast cancer

PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients. Several of those patients exhibit intrinsic/acquired resistance mechanisms that limit efficacy of PARPi monotherapy. Here we show how the efficacy of PARPi in triple-negative breast cancers (TNBC) can be expanded by targeting MYC-induced oncogenic addiction. In BRCA-mutant/sporadic TNBC patients, amplification of the MYC gene is correlated with increased expression of the homologous DNA recombination enzyme RAD51 and tumors overexpressing both genes are associated with worse overall survival. Combining MYC blockade with PARPi yielded synthetic lethality in MYC-driven TNBC cells. Using the cyclin-dependent kinase inhibitor dinaciclib, which downregulates MYC expression, we found that combination with the PARPi niraparib increased DNA damage and downregulated homologous recombination, leading to subsequent downregulation of the epithelial-mesenchymal transition and cancer stem-like cell phenotypes. Notably, dinaciclib resensitized TBNC cells, which had acquired resistance to niraparib. We found that the synthetic lethal strategy employing dinaciclib and niraparib was also highly efficacious in ovarian, prostate, pancreatic, colon, and lung cancer cells. Taken together, our results show how blunting MYC oncogene addiction can leverage cancer cell sensitivity to PARPi, facilitating the clinical use of c-myc as a predictive biomarker for this treatment.Significance: Dual targeting of MYC-regulated homologous recombination and PARP-mediated DNA repair yields potent synthetic lethality in triple-negative breast tumors and other aggressive tumors characterized by MYC overexpression. Cancer Res; 78(3); 742-57. ©2017 AACR.

Low-Molecular-Weight Cyclin E in Human Cancer: Cellular Consequences and Opportunities for Targeted Therapies

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (CDK2), is central to the initiation of DNA replication at the G1/S checkpoint. Tight temporal control of cyclin E is essential to the coordination of cell-cycle processes and the maintenance of genome integrity. Overexpression of cyclin E in human tumors was first observed in the 1990s and led to the identification of oncogenic roles for deregulated cyclin E in experimental models. A decade later, low-molecular-weight cyclin E (LMW-E) isoforms were observed in aggressive tumor subtypes. Compared with full-length cyclin E, LMW-E hyperactivates CDK2 through increased complex stability and resistance to the endogenous inhibitors p21CIP1 and p27KIP1 LMW-E is predominantly generated by neutrophil elastase-mediated proteolytic cleavage, which eliminates the N-terminal cyclin E nuclear localization signal and promotes cyclin Es accumulation in the cytoplasm. Compared with full-length cyclin E, the aberrant localization and unique stereochemistry of LMW-E dramatically alters the substrate specificity and selectivity of CDK2, increasing tumorigenicity in experimental models. Cytoplasmic LMW-E, which can be assessed by IHC, is prognostic of poor survival and predicts resistance to standard therapies in patients with cancer. These patients may benefit from therapeutic modalities targeting the altered biochemistry of LMW-E or its associated vulnerabilities. Cancer Res; 78(19); 5481-91. ©2018 AACR.

Abstract 2060: Characterizing acquired resistance to palbociclib in breast cancer

The CDK4/6 inhibitor palbociclib is currently being used in combination with endocrine therapy to treat advanced ER positive breast cancer patients. While this treatment has shown great promise in the clinic, about 25% of the patients do not respond, and almost all patients eventually acquire resistance to palbociclib treatment. Hence, understanding the mechanism(s) of acquired resistance to CDK4/6 inhibition is crucial to devise alternate treatment strategies. To interrogate this, we developed MCF7 and T47D resistant cells by treating them with increasing doses of palbociclib over a 6-month period. After confirming that these cells were resistant to palbociclib, we performed genome-wide expression analysis via RNA-seq, in comparison with the parental (sensitive) cells. RNA- seq analysis revealed 2888 differentially expressed genes (p Citation Format: Smruthi Vijayaraghavan, Iman Doostan, Jason P.W. Carey, Khandan Keyomarsi. Characterizing acquired resistance to palbociclib in breast 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 2060. doi:10.1158/1538-7445.AM2017-2060

Abstract B27: CDK inhibition impairs homologous recombination and induces PARP inhibitor sensitivity via loss of c-myc expression in TNBC

The c-myc oncogene is a “master regulator” amplified in several types of cancer including ovarian, breast, colorectal and pancreatic cancers. C-myc controls several cellular processes during tumor maintenance including the cell cycle, metabolism, and DNA damage repair while also playing an essential role in transforming normal cells during tumorigensis. C-myc amplification correlates with poor survival and a higher frequency of relapse in triple negative breast cancer (TNBC) patients. C-myc amplification promotes deregulation of the cell cycle, via transcriptional activation of G1-S-phase cell cycle and DNA replication genes. CDK inhibition has been shown to down regulate c-myc expression in cancer cells, in vitro. Therefore, we hypothesize that transient CDK inhibition downregulates c-myc expression and increases susceptibility to DNA damaging agents in TNBC cells. We observed the long-term response of several c-myc overexpressing TNBC cells to Dinaciclib, a potent CDK inhibitor (CDK 1, 2, 5, 9) in vitro. High Throughput Survival Assay (HTSA) analysis revealed that c-myc expressing TNBC cells have a potent response to CDK inhibition via Dinaciclib (IC50 10-20nM). Dinaciclib induces an S-phase arrest and downregulation of the c-myc/E2F1 pathway in both normal (MCF10A) and TNBC (MB231, SUM149, HCC1937) breast cancer cells. Loss of c-myc expression correlates with increased apoptosis in TNBC cells but not HMECs. C-myc downregulation also correlates with increased γH2AX DNA damage and downregulation of BRCA1 foci formation. The resultant impaired homologous recombination (HR) pathway in TNBC cells treated with Dinaciclib proposed an increased susceptibility to PARP inhibitor induced DNA damage. Combination Dinaciclib + PARP inhibition exhibited a Dinaciclib dose dependent synergistic growth inhibition of BRCA1 wild-type and mutant TNBC cells to PARP inhibitors. Dinaciclib also increased susceptibility of PARP inhibition in resistant BRCA1 mutant cell lines (HCC1937, SUM149). Dinaciclib + PARP Inhibitor combination therapy induced increased apoptosis, γH2AX DNA damage and decreased BRCA1, RAD51 foci formation. These results demonstrate that Dinaciclib + PARP inhibition induces growth arrest and induction of apoptosis in c-myc overexpressing cancer cells. C-myc is frequently amplified in TNBC tumors and may serve as a biomarker for CDK + PARP inhibitor combination therapy. Citation Format: Jason Patrick Carey, Khandan Keyomarsi. CDK inhibition impairs homologous recombination and induces PARP inhibitor sensitivity via loss of c-myc expression in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B27.

Abstract P6-03-09: Targeting the c-myc/E2F1 pathway in TNBC promotes a DNA damage dependent synthetic lethality

The c-myc/E2F1 pathway is highly upregulated in TNBC and dictates increased genomic instability, a higher frequency of relapse and poor overall survival. C-myc is a potent oncogene with overexpression activity that influences several pathways including metabolism, DNA replication, DNA damage, cell cycle and apoptosis. The essential role of c-myc in normal cells has made therapeutic intervention elusive. However the role of c-myc influencing cell cycle progression has demonstrated that CDK inhibition downregulates c-myc expression in TNBC breast cancer cells. We hypothesize that CDK inhibition downregulates c-myc expression resulting in an accumulation of DNA damage thus increasing the susceptibility of TNBC cells to PARP inhibition. A panel of TNBC cell lines was assessed for c-myc/E2F1 pathway expression and downregulation in the presence of CDK inhibitor Dinaciclib. An MTT-based High Throughput Survival Assay (HTSA) was utilized to assess growth inhibition response to drug treatment in TNBC cells. FACS analysis was performed on Dinaciclib treated TNBC cell lines to asses cell cycle response to CDK inhibition. DNA damage and Homologous Recombination DNA repair was assessed by γH2AX, BRCA1, RAD51 foci formation by immunofluorescence. Lentiviral knockdown of c-myc, E2F1, BRCA1 and RAD51 were performed to assess susceptibility to PARP inhibition. Combination treatment of Dinaciclib + PARP inhibitor MK4827 was assessed via combination index values calculated by Calcusyn. CDK inhibition resulted in downregulation of both c-myc and E2F1 in all TNBC cells. The pan-CDK inhibitor Dinaciclib universally inhibited proliferation in all TNBC cell lines in the presence of the drug however drug removal allowed TNBC cells to recover a normal proliferative profile. Dinaciclib induction increased γH2AX foci formation while decreasing BRCA1 and RAD51 foci formation resulting in an increase in DNA damage induced cell death that correlated with c-myc downregulation. CDK inhibition correlated with downregulation of c-myc/E2F1 target genes. Lentiviral knockdown of c-myc and not E2F1 induced susceptibility to PARP inhibitor MK4827. Combination CDK + PARP inhibitor resulted in a dose dependent synthetic lethal increase in PARP inhibitor efficacy in both BRCA1 mutant and wild-type cells. Together these results suggest that c-myc downregulation via CDK inhibition in combination with PARP inhibition may pose a novel combination therapy for TNBC patients. Citation Format: Jason P Carey, Khandan Keyomarsi. Targeting the c-myc/E2F1 pathway in TNBC promotes a DNA damage dependent synthetic lethality [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-09.

Abstract 1327: Targeting the RB-pathway in sarcoma: Utility of CDK4/6 inhibitors

Background: Sarcomas are rare neoplasms of mesenchymal origin. Survival rates have not improved with standard chemotherapy; however, molecular insights have posed new drug targets. Aberrations in the RB-pathway, including loss of RB, CDK4 and cyclin D1 overexpression and p16 loss, have been identified in sarcomas. PD 03322991 (PD) is a second generation CDK4/6 inhibitor with the potential to reactivate the RB-pathway and suppress cell cycle progression. We evaluated the efficacy of PD in a panel of sarcoma cell lines. Methods: Sarcoma cell lines (HT-1080, SK-LMS-1 and SaOS-2) were evaluated for RB-pathway alterations using western blot. Cells were treated with PD for 1, 2, 4, 6 and 8 days to determine IC50 values by MTT assay. Following the same treatment protocol at IC50 concentrations of PD, cells were harvested for cell cycle analysis, western blot and annexin cell death assay. Results: HT-1080 and SK-LMS-1 were RB-positive with high expression of phospho-RB; SaOS-2 was RB-negative. CDK4/6 were uniformly expressed. HT-1080 and SK-LMS-1 had p16 loss while SaOS-2 overexpressed cyclin D1. PD was effective in all cell lines. Increasing treatment duration led to reduction of IC50 values. PD caused marked G1 arrest as early as day 1 in RB-positive HT-1080 (86.2% vs 74.2% [vehicle]) and SK-LMS-1 (91.4% vs 67.8% [vehicle]) but not RB-negative SaOS-2 (52.9-59.4% vs 57.0% [vehicle]). Annexin assay demonstrated minimal apoptosis with PD (HT-1080: 1.4-3.0% vs 41.2% [control], SK-LMS-1: 0.6-12.0% vs 23.4% [control], SaOS-2: 3.6-7.3% vs 78.7% [control]). Conclusions: PD appears to have a cytostatic rather than cytotoxic effect in sarcoma. RB-negativity does not preclude PD efficacy but may require higher IC50 values. In RB-negative sarcomas, PD does not cause cell cycle arrest suggesting an alternate mechanism from reactivation of the RB-pathway is present. PD may serve as a potential therapy for sarcoma and should be studied in combination with other targeted therapies that can take advantage of its ability to cause G1 arrest. Citation Format: Ashleigh M. Francis, Jason P. Carey, Angela Alexander, Khandan Keyomarsi, Kelly K. Hunt. Targeting the RB-pathway in sarcoma: Utility of CDK4/6 inhibitors. [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 1327. doi:10.1158/1538-7445.AM2014-1327