Karnitz, Larry M.

Abstract The human ATR gene encodes a kinase that is activated by DNA damage and replication stress as a central transducer of a checkpoint signaling pathway. Once activated, ATR phosphorylates multiple substrates, including the kinase Chk1, to regulate cell-cycle progression, replication fork stability, and DNA repair. These events promote cell survival during replication stress and in cells with DNA damage. Accordingly, there has been the tantalizing possibility that ATR inhibitors would be therapeutically useful, especially if they were more effective in tumor versus normal cells. Indeed, multiple studies have demonstrated that alterations that promote tumorigenesis, such as defects in the ATM-p53 pathway, constitutive oncogene activation, and acquisition of the alternative lengthening of telomeres pathway, render tumor cells sensitive to ATR inhibitor monotherapy and/or increase the synergy between ATR inhibitors and genotoxic chemotherapies. Now, nearly two decades after the discovery of ATR, two highly selective and potent ATR inhibitors, AZD6738 and VX-970, are in early-phase clinical trials either as monotherapies or paired with a variety of genotoxic chemotherapies. These trials will generate important insights into the effects of ATR inhibition in humans and the potential role of inhibiting this kinase in the treatment of human malignancies. Clin Cancer Res; 21(21); 4780–5. ©2015 AACR.

Abstract Purpose: Treatment of recurrent ovarian cancer is limited by low response rates to salvage chemotherapy. Novel small molecule inhibitors to Chk1 (checkpoint kinase 1) have been proposed to potentiate the cytotoxic effect of one such salvage treatment, gemcitabine, by interrupting the DNA damage repair pathway. Patient derived tumorgrafts were characterized by responsiveness to gemcitabine +/- MK8776 (Chk1 inhibitor). Response was correlated with markers of Chk1 activity. Methods: Patient-derived tumorgraft models were created from patients undergoing surgery for epithelial ovarian cancer. Gemcitabine response was characterized in five high-grade serous models (PH044, PH048, PH070, PH080, and PH110). Two models (PH048 and PH080) were treated with gemcitabine monotherapy (240 mg/kg qweek x 4 doses), MK8776 monotherapy (32 mg/kg qweek x 4 doses), and with combination therapy (Gemcitabine 190 mg/kg and MK8776 32 mg/kg qweek x 4 doses). Animals were monitored for cytotoxicity, and tumors were measured weekly with ultrasound. Response to gemcitabine and to combination therapy was correlated to immunohistochemistry of γH2AX and immunoblotting for p345-Chk1 and p296-Chk1. Results: Gemcitabine monotherapy did not affect tumor growth in two models (PH044 and PH048). Significant in vivo response to gemcitabine was seen in PH070 and PH080. Significant growth attenuation but not tumor regression was seen in PH110. MK8776 did not have any effect on tumor growth as monotherapy or in combination with gemcitabine in the gemcitabine resistant model, PH048. In contrast, in a gemcitabine sensitive model, PH080, MK8776 monotherapy significantly attenuated tumor growth, and there was a trend for MK8776 to potentiate the cytotoxic effects of gemcitabine in PH080 (p=0.16). Final tumor weights in the PH080 model after 4 doses of chemotherapy were 1.7 g (90%CI: 1.5-1.9g), 0.3g (90%CI: 0.18-0.35g), and 0.14g (90%CI: 0.08-.19g) in the control, gemcitabine alone, and gemcitabine with MK8776 groups respectively. Decrease in the tumor auto-phosphorylation of Chk1 at Serine-296 was observed in some, but not all models in response to MK8766 treatment. γH2AX increased in response to the addition of MK8776 to gemcitabine. Conclusions: Chk1 inhibition may enhance the activity of gemcitabine in some, but not all ovarian tumors. Further work is needed to find markers that will predict response to Chk1 inhibitors to help select patients for future clinical trials. While pChk1-S296 previously demonstrated potential utility as a biomarker of Chk1 inhibition, in vivo assessments of its differential expression may demonstrate the unreliability of this marker. This work supports the use of patient derived tumorgrafts in drug development for ovarian cancer. Citation Format: A Kumar, S Weroha, M Becker, X Hou, S Greiner, L Karnitz, P Haluska. Chk1 inhibitor (MK8776) enhances cytotoxicity of gemcitabine in select patient-derived ovarian tumorgraft models [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1419.

Abstract PURPOSE: The purpose of this study was to determine whether a) high-grade serous ovarian cancer-associated CDK12 kinase domain mutations affect catalytic activity, b) loss of CDK12 affects BRCA1 expression levels and Homologous Recombination (HR) in ovarian cancer, c) tumor-associated CDK12 mutations cause HR defects in ovarian cancer, and d) loss of CDK12 sensitizes ovarian cancer to chemotherapy and Poly(ADP-ribose) Polymerase [PARP] inhibitors. EXPERIMENTAL PROCEDURES: We compared the kinase activities of wild-type CDK12 and tumor-associated CDK12 kinase domain mutants using in-vitro kinase assays. To determine the impact of CDK12 activity on HR, we assessed RAD51 foci formation and HR using a DR-GFP construct. To analyze the effect of tumor-associated CDK12 mutations on HR, CDK12-depleted OVCAR-8 cells were reconstituted with either wild-type CDK12 or the tumor-associated CDK12 mutant (G909R), irradiated and stained for RAD51 foci. We performed clonogenic assays using ovarian cancer cell lines (OVCAR-8, OVCAR-5 and OVCAR-3) to measure sensitivity to DNA cross-linking agents (melphalan and cisplatin) and the PARP inhibitor, veliparib, in the presence or absence of CDK12 activity. CDK12 depletion was carried out using at least two independent short-interfering RNAs (siRNAs). RESULTS: We showed that many, but not all, ovarian cancer-associated CDK12 kinase domain mutations severely disrupted kinase activity. Moreover, we found that disabling CDK12 activity in ovarian cancer cells decreased BRCA1 levels and disrupted HR. Additionally, the tumor-associated CDK12 G909R mutant did not restore impaired RAD51 foci formation in CDK12-depleted OVCAR-8 cells. Finally we show that CDK12 depletion sensitized multiple ovarian cancer cells to the DNA cross-linking agents melphalan and cisplatin and to the PARP inhibitor veliparib. CONCLUSION: Ovarian cancer-associated CDK12 mutations disrupt kinase activity and cause defects in HR, suggesting that ovarian tumors harboring deleterious CDK12 mutations may predict sensitivity to chemotherapeutic agents, including PARP inhibitors. Citation Format: Poorval M. Joshi, Shari L. Sutor, Catherine J. Huntoon, Larry M. Karnitz. Ovarian cancer-associated CDK12 mutations disable its kinase activity and cause defects in homologous recombination [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1325.

Abstract Background: Cytarabine and an anthracycline, the mainstay of AML therapy, affords a five-year survival below 30%. Accordingly, there is a continuing need for improved AML therapies. CPX-351 encapsulates within liposomes a fixed 5:1 molar ratio of cytarabine and daunorubicin that is synergistic (in vitro) and markedly improved in anti-leukemic activity (in vivo). Promising evidence of improved remission and survival rates with CPX-351 compared to standard of care treatments has also been observed clinically and a pivotal Phase 3 trial is currently ongoing. Because Chk1 inhibition has been shown to enhance the actions of both cytarabine and anthracyclines, there has been interest in assessing the effect of CPX-351 when combined with Chk1 inhibitors. Here we describe the evaluation of CPX-351 alone and in combination with the Chk1 inhibitors MK-8776 and LY2603618 in AML cell lines and in clinical AML samples ex vivo. Methods: Effects of CPX-351 on Chk1 phosphorylation and cell cycle distribution in the human AML lines U937 and HL-60 were analyzed by immunoblotting and flow cytometry. Effects of Chk1 inhibitors on CPX-351-induced apoptosis were assessed by flow cytometry after propidium iodide staining (DNA cleavage), Hoechst staining (nuclear fragmentation) and colony formation in soft agar (long-term survival). Effects CPX-351 ± MK-8776 on clinical AML samples were assayed using clonogenic assays. Results: Treatment of U937 cells with CPX-351 resulted in phosphorylation of Chk1 on Ser317 and Ser296 (sites of ATR-mediated phosphorylation and autophosphorylation, respectively) as well as S phase slowing consistent with Chk1 activation. Co-treatment of AML cell lines with Chk1 inhibitors + CPX-351 resulted in increased apoptotic morphological changes and DNA fragmentation as well as diminished clonogenic survival compared to CPX-351 alone. The CPX-351/MK-8776 combination displayed enhanced antiproliferative effects against some clinical AML isolates ex vivo compared to CPX-351 alone, with additional assays ongoing. Conclusions: Collectively, these results show that Chk1 inhibition enhances the cytotoxicity of CPX-351 in AML cell lines and some clinical AML samples, offering the possibility that CPX-351 may be well suited for combining with such molecularly targeted agents. Citation Format: Nicole D. Vincelette, Larry M. Karnitz, Judith E. Karp, Douglas B. Smith, Allan D. Hess, Lawrence D. Mayer, Scott H. Kaufmann. CPX-351 (cytarabine:daunorubicin liposome for injection) anti-leukemia activity is potentiated by Chk1 inhibition. [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 3479. doi:10.1158/1538-7445.AM2015-3479

Abstract Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)–mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells. Cancer Res; 73(12); 3683–91. ©2013 AACR.

Abstract Purpose: Previous studies have shown that the replication checkpoint, which involves the kinases ataxia telangiectasia mutated and Rad3 related (ATR) and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical acute myelogenous leukemia (AML) during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design: AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA, and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results: Immunoblotting revealed increased Chk1 phosphorylation, a marker of checkpoint activation, in more than half of Chk1-containing AMLs after 48 hours of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S-phase arrest but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the antiproliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions: These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted. Clin Cancer Res; 18(19); 5364–73. ©2012 AACR.

Abstract 5-Fluorouracil (5-FU) and 5-fluorodeoxyuridine (FdUrd, floxuridine) have activity in multiple tumors, and both agents undergo intracellular processing to active metabolites that disrupt RNA and DNA metabolism. These agents cause imbalances in deoxynucleotide triphosphate levels and the accumulation of uracil and 5-FU in the genome, events that activate the ATR- and ATM-dependent checkpoint signaling pathways and the base excision repair (BER) pathway. Here, we assessed which DNA damage response and repair processes influence 5-FU and FdUrd toxicity in ovarian cancer cells. These studies revealed that disabling the ATM, ATR, or BER pathways using small inhibitory RNAs did not affect 5-FU cytotoxicity. In stark contrast, ATR and a functional BER pathway protected FdUrd-treated cells. Consistent with a role for the BER pathway, the poly(ADP-ribose) polymerase (PARP) inhibitors ABT-888 (veliparib) and AZD2281 (olaparib) markedly synergized with FdUrd but not with 5-FU in ovarian cancer cell lines. Furthermore, ABT-888 synergized with FdUrd far more effectively than other agents commonly used to treat ovarian cancer. These findings underscore differences in the cytotoxic mechanisms of 5-FU and FdUrd and suggest that combining FdUrd and PARP inhibitors may be an innovative therapeutic strategy for ovarian tumors. Cancer Res; 71(14); 4944–54. ©2011 AACR.

The Fanconi anemia (FA) network is important for the repair of interstrand DNA cross-links. A key event in FA pathway activation is the monoubiquitylation of the FA complementation group I (FANCI)–FANCD2 (ID) complex by FA complementation group L (FANCL), an E3 ubiquitin ligase. In this study, we show that RAD18, another DNA damage–activated E3 ubiquitin ligase, also participates in ID complex activation by ubiquitylating proliferating cell nuclear antigen (PCNA) on Lys164, an event required for the recruitment of FANCL to chromatin. We also found that monoubiquitylated PCNA stimulates FANCL-catalyzed FANCD2 and FANCI monoubiquitylation. Collectively, these experiments identify RAD18-mediated PCNA monoubiquitination as a central hub for the mobilization of the FA pathway by promoting FANCL-mediated FANCD2 monoubiquitylation.

Abstract Heat shock protein 90 (HSP90), which regulates the functions of multiple oncogenic signaling pathways, has emerged as a novel anticancer therapeutic target, and multiple small-molecule HSP90 inhibitors are now in clinical trials. Although the effects of HSP90 inhibitors on oncogenic signaling pathways have been extensively studied, the effects of these agents on tumor suppressor signaling pathways are currently unknown. Here, we have examined how HSP90 inhibitors affect LATS1 and the related protein LATS2, two kinases that relay antiproliferative signals in the Hippo tumor suppressor pathway. Both LATS1 and LATS2 were depleted from cells treated with the HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG), radicicol, and PU-H71. Moreover, these kinases interacted with HSP90, and LATS1 isolated from 17-AAG–treated cells had reduced catalytic activity, thus showing that the kinase is a bona fide HSP90 client. Importantly, LATS1 signaling was disrupted by 17-AAG in tumor cell lines in vitro and clinical ovarian cancers in vivo as shown by reduced levels of LATS1 and decreased phosphorylation of the LATS substrate YAP, an oncoprotein transcriptional coactivator that regulates genes involved in cell and tissue growth, including the CTGF gene. Consistent with the reduced YAP phosphorylation, there were increased levels of CTGF, a secreted protein that is implicated in tumor proliferation, metastasis, and angiogenesis. Taken together, these results identify LATS1 and LATS2 as novel HSP90 clients and show that HSP90 inhibitors can disrupt the LATS tumor suppressor pathway in human cancer cells. Cancer Res; 70(21); 8642–50. ©2010 AACR.

The Sonic Hedgehog (Shh) pathway plays important roles in embryogenesis, stem cell maintenance, tissue repair, and tumorigenesis. Haploinsufficiency of Patched-1, a gene that encodes a repressor of the Shh pathway, dysregulates the Shh pathway and increases genomic instability and the development of spontaneous and ionizing radiation (IR)–induced tumors by an unknown mechanism. Here we show that Ptc1+/− mice have a defect in the IR-induced activation of the ATR–Chk1 checkpoint signaling pathway. Likewise, transient expression of Gli1, a downstream target of Shh signaling, disrupts Chk1 activation in human cells by preventing the interaction of Chk1 with Claspin, a Chk1 adaptor protein that is required for Chk1 activation. These results suggest that inappropriate Shh pathway activation promotes tumorigenesis by disabling a key signaling pathway that helps maintain genomic stability and inhibits tumorigenesis.