Combining Chk1 Inhibitors Research Articles

Targeting the cell cycle in head and neck cancer by Chk1 inhibition: a novel concept of bimodal cell death

Head and neck squamous cell carcinomas (HNSCCs) coincide with poor survival rates. The lack of driver oncogenes complicates the development of targeted treatments for HNSCC. Here, we follow-up on two previous genome-wide RNA and microRNA interference screens in HNSCC to cross-examine tumor-specific lethality by targeting ATM, ATR, CHEK1, or CHEK2. Our results uncover CHEK1 as the most promising target for HNSCC. CHEK1 expression is essential across a panel of HNSCC cell lines but redundant for growth and survival of untransformed oral keratinocytes and fibroblasts. LY2603618 (Rabusertib), which specifically targets Chk1 kinase, kills HNSCC cells effectively and specifically. Our findings show that HNSCC cells depend on Chk1-mediated signaling to progress through S-phase successfully. Chk1 inhibition coincides with stalled DNA replication, replication fork collapses, and accumulation of DNA damage. We further show that Chk1 inhibition leads to bimodal HNSCC cell killing. In the most sensitive cell lines, apoptosis is induced in S-phase, whereas more resistant cell lines manage to bypass replication-associated apoptosis, but accumulate chromosomal breaks that become lethal in subsequent mitosis. Interestingly, CDK1 expression correlates with treatment outcome. Moreover, sensitivity to Chk1 inhibition requires functional CDK1 and CDK4/6 to drive cell cycle progression, arguing against combining Chk1 inhibitors with CDK inhibitors. In contrast, Wee1 inhibitor Adavosertib progresses the cell cycle and thereby increases lethality to Chk1 inhibition in HNSCC cell lines. We conclude that Chk1 has become a key molecule in HNSCC cell cycle regulation and a very promising therapeutic target. Chk1 inhibition leads to S-phase apoptosis or death in mitosis. We provide a potential efficacy biomarker and combination therapy to follow-up in clinical setting.

Abstract 1608: Pharmacologic screening identifies synthetic lethality interactions with Chk1 inhibitors in basal-like breast and ovarian cancer

Abstract Basal-like breast cancer and ovarian cancer are considered as unmet entities as patient outcome in advanced stages is short, even receiving the most appropriate treatment. These tumors are characterized by a high grade of genetic instability and a notable impairment of DNA repair processes. Thus, in response to DNA damage, they depend on checkpoint arrest proteins, such as Chek1, to stop division. Inactivation of Chk1 permits cell cycle progression without a proper DNA repair, therefore increasing genetic instability and aneuploidy and eventually leading to cell death. Chk1 inhibitors are currently in clinical development and, therefore, the identification of chemotherapies that could be used as clinical partners to potentiate their effect is a main goal. OVCAR3 and IGROV ovarian cancer cells as well as the basal-like MDA-MB-231 and HS578T cell lines were used to screen for agents that may increase the antiproliferative properties of Chk1 inhibitors. Combination of DNA-damaging agents, like platinum compounds or gemcitabine, with the Chk1 inhibitors LY2603618 and SAR020106 had a synergistic effect, not observed when combined with agents targeting mitosis, like taxotere, eribulin or vinorelbine. A similar antiproliferative effect was observed for PARP inhibitors such as olaparib, reinforcing the concept that inhibition of Chk1 allows cells to enter apoptosis when a relevant genetic instability is present. Clonogenic assays and 3D colony formation studies confirmed the effect observed on proliferation also on invasion and long-term survival. Analyses of cell cycle, cell death and intracellular mediators were performed by flow cytometry and Western blot. The effect of the association of LY2603618 with cisplatin and gemcitabine did not change the cell cycle. However, combination of both agents produced a marked induction of apoptosis. Experiments evaluating caspase activity demonstrated that the principal mechanism associated with the induction of apoptosis was caspase dependent. We also explored combination of agents that have a clear translation to the clinics including double combinations with chemotherapies like cisplatin and gemcitabine. We observed that these triple combinations were more effective than double or single agents. Finally, we wanted to evaluate the antiproliferative effect when adding LY2603618 to olaparib in addition to a DNA-damaging agent. Of note, we observed a higher effect than single-agent olaparib or the chemotherapy. Interestingly, inhibition of Chk1 also reverted the effect of cisplatin in platinum-resistant cells. Our findings support the clinical development of Chk1 inhibitors in combination with DNA-damaging agents in basal-like and ovarian cancer. Citation Format: Eva M. Galan-Moya, Ana Alcaraz-Sanabria, Cristina Nieto-Jimenez, Veronica Corrales-Sanchez, Miriam Nuncia-Cantarero, Juan C. Montero, Atanasio Pandiella, Alberto Ocaña. Pharmacologic screening identifies synthetic lethality interactions with Chk1 inhibitors in basal-like breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1608.

MYC regulated long non-coding RNAs could have roles in development of breast cancer

The deregulation of the cell cycle and checkpoint machinery in cancer presents a highly attractive therapeutic strategy. We review here the strategies used to exploit the dysregulated cell cycle, both through targeting kinases required for cell cycle progression, and checkpoint kinases to inappropriately force cells through the cell cycle. Appropriate control of the cell cycle is critical for proliferating normal cells, and we discuss the importance of defining tumour specific vulnerabilities that could be targeted with cell cycle kinase inhibitors. Recent studies have shown that ER-positive breast cancers rely on CDK4 to promote proliferation. TP53 mutant cancer cell lines are sensitive to WEE1 and CHK1 inhibitors in combination with chemotherapy, while PTEN-deficient aneuploid cancer cell lines are sensitive to TTK inhibitors.

Cell cycle perturbation induced by gemcitabine in human tumor cells in cell culture, xenografts and bladder cancer patients: implications for clinical trial designs combining gemcitabine with a Chk1 inhibitor.

Gemcitabine irreversibly inhibits ribonucleotide reductase and induces S phase arrest but whether this occurs in tumors in mice or patients has not been established. Tumor cells in culture were incubated with gemcitabine for 6 h to approximate the administration schedule in a patient. Concentrations that induced persistent S phase arrest thereafter correlated with cell killing. Administration of gemcitabine to mice also demonstrated a persistent S phase arrest in their tumor. The minimum dose that induced almost complete S phase arrest after 24 h (40 mg/kg) was well below the maximum tolerated dose in mice. S phase arrest was also observed in tumors of bladder cancer patients receiving gemcitabine. The Chk1 inhibitor MK-8776 sensitized cells to gemcitabine with the greatest cell killing when added 18 h after gemcitabine. In mice, the administration of MK-8776 18 h after gemcitabine elicited positivity for the DNA damage marker γH2AX; this also occurred at relatively low dose (40 mg/kg) gemcitabine. Hence, in both cell culture and xenografts, MK-8776 can markedly enhance cell killing of cells reversibly arrested in S phase by gemcitabine. Some cell lines are hypersensitive to MK-8776 as monotherapy, but this was not observed in xenograft models. Effective monotherapy requires a higher dose of Chk1 inhibitor, and target inhibition over a longer time period as compared to its use in combination. These results have important implications for combining Chk1 inhibitors with gemcitabine and suggest that Chk1 inhibitors with increased bioavailability may have improved efficacy both in combination and as monotherapy.

Abstract IA13: CHK1 inhibitors: From concept to clinic

Abstract Each cell in the human body is subjected daily to both exogenous and endogenous insults that result in DNA damage. This assault on the human genome can lead to the formation of many different types of DNA lesions such as single strand breaks (SSBs), double strand breaks (DSBs), base modifications and base mismatches that are repaired by a number of distinct but interrelated mechanisms. However, the failure to detect and repair these lesions can lead to cells with intrinsically high levels of DNA damage, gene mutations and often even larger genomic changes including chromosomal aberrations. These changes within the DNA may all contribute to the genome becoming unstable, such that there is an elevated frequency of genomic alterations. This genomic instability has now been acknowledged as a hallmark of cancer. Detection and response to DNA Damage is therefore essential for cells and is undertaken by the DNA damage response (DDR) pathway, an intracellular signaling network that coordinates and regulates the cellular activities that can detect and repair damaged DNA. A key component of this is pathway is the serine/threonine kinase CHK1. This kinase has the ability, through substrate phosphorylation to halt the cell division cycle, in particular at S and G2/M checkpoints. This gives the cell time to either repair the damaged DNA and re-initiate the cell cycle or if effective DNA repair is not possible, to induce cell senescence or cell death. The DDR is important for both normal and cancer cells. Cancer cells however, often have gene alterations that cause defects in one or more cell cycle checkpoints or in DNA repair. An important gene in this context is TP53 which encodes the p53 protein that can activate the G1/S cell cycle checkpoint in response to DNA damage. A hallmark of many tumors is the lack of functional p53 protein with a consequent loss of the G1/S checkpoint leading to a potential increased reliance on the S and G2/M checkpoints for survival. It was therefore initially hypothesized that CHK1 inhibitors may selectively enhance the activity of genotoxic agents in tumors, in particular by abrogating the G2 checkpoint, while normal cells would be rescued due to their competent DDR. This led to development a number of CHK1 inhibitors that are progressing towards, or are currently undergoing, clinical evaluation in combination with specific genotoxic cancer drugs. These include the potent, selective and orally bioavailable CHK1 inhibitor CCT245737, which is now scheduled for Phase I clinical trial and will be discussed during this presentation. Research now suggests that the role of CHK1 during S-phase may also be important when combining CHK1 inhibitors with selected chemotherapeutic agents and this will also be discussed. A key role for CHK1 during S phase is upon replication stress, where it stabilizes and preserves replication fork complexes thus preventing catastrophic replication fork collapse, until replication can be re-initiated. As well as suffering from genome instability, many cancer cells exhibit high levels of replication stress, which is thought to be due to oncogene-driven proliferation. Therefore tumors with high levels of replication stress will be dependent on functional CHK1. This provides a potential monotherapy strategy for CHK1 inhibitors in selected adult and pediatric cancer settings that are oncogene-driven and/or exhibit replication stress, which will be discussed in this presentation. Citation Format: Michelle D. Garrett. CHK1 inhibitors: From concept to clinic. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA13.

Abstract P4-07-02: Targeted therapies for TNBC: Exploiting vulnerabilities that arise from DNA damage repair pathway dependencies

Abstract We examined the synergistic effects of DNA damage, Chk1 inhibition and poly(ADP-ribose) polymerase (PARP) inhibition in TNBC. This combinatorial targeting allows us to exploit vulnerabilities in two pathways that are often deregulated in TNBCs: DNA damage checkpoint defects due to TP53 deficiency and DNA repair defects due to alterations in homologous recombination repair (HRR). TP53 maintains genome integrity by inhibiting cells that are experiencing genotoxic stress from progressing through the cell cycle, or by inducing apoptosis or senescence. In response to DNA damage, p53 activates gene expression to arrest cells in the G1 phase of the cell cycle and to reinforce the S- and G2-checkpoints. Thus, p53-deficient cells lack a G1 checkpoint and are impaired in their ability to sustain S- and G2-checkpoints. This makes p53-deficient tumors particularly sensitive to agents that abrogate these checkpoints. Because Chk1 inhibitors abrogate both S- and G2-checkpoints, combining Chk1 inhibitors with agents that induce genotoxic stress provides a rational therapeutic strategy for killing p53-deficient TNBC. Loss of HRR increases dependence of cells on a class of enzymes called PARPs, and Chk1 has also been shown to be important for efficient HRR. Thus, by interfering with HRR, Chk1 inhibitors are predicted to sensitize TNBC cells to PARP inhibitors. We tested the hypotheses that by impairing HRR, Chk1 inhibitors will sensitize TNBCs to PARP inhibition, and that therapies that combine Chk1 inhibitors with PARP inhibitors will be effective at killing TNBCs because they will simultaneously induce checkpoint bypass and block DNA repair. We generated a set of isogenic TNBC cell lines that are p53-proficient (p53WT) or p53-deficient (p53KD), and evaluated their sensitivity to Chk1 inhibitors (LY2606368) and DNA damaging agents (cisplatin). Loss of p53 conferred a dramatic increase in sensitivity to treatment with cisplatin + LY2606368. Surprisingly, inhibition of PARP1 (BMN673) did not increase sensitivity to Chk1 inhibitor ± cisplatin. To determine why Chk1 inhibition did not sensitize cells to PARP inhibition, we evaluated the effect of Chk1 inhibition on the ability of cells to recruit HRR proteins to sites of DNA damage. In line with CHK1 regulating HRR, Chk1 inhibition was associated with an inability of Rad51 to localize to sites of DNA double strand breaks. Interestingly, we also found that upstream of Rad51, there was a significant alteration in the formation of phopho-RPA2 foci in cells treated with the Chk1 inhibitor. On-going studies are evaluating whether there are changes in the kinetics of formation and/or resolution of Rad51 and phospho-RPA2 foci in response to Chk1 inhibition. Citation Format: Redwood AB, Cai S, Piwnica-Worms H. Targeted therapies for TNBC: Exploiting vulnerabilities that arise from DNA damage repair pathway dependencies. [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 P4-07-02.

Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition.

BackgroundThe hypoxia-activated prodrug TH-302 is reduced at its nitroimidazole group and selectively under hypoxic conditions releases the DNA cross-linker bromo-isophosphoramide mustard (Br-IPM). Here, we have explored the effect of Chk1 inhibition on TH-302-mediated pharmacological activities.MethodsWe employed in vitro cell viability, DNA damage, cellular signaling assays and the in vivo HT29 human tumor xenograft model to study the effect of Chk1inhibition on TH-302 antitumor activities.ResultsTH-302 cytotoxicity is greatly enhanced by Chk1 inhibition in p53-deficient but not in p53-proficient human cancer cell lines. Chk1 inhibitors reduced TH-302-induced cell cycle arrest via blocking TH-302-induced decrease of phosphorylation of histone H3 and increasing Cdc2-Y15 phosphorylation. Employing the single-cell gel electrophoresis (comet) assay, we observed a potentiation of the TH-302 dependent tail moment. TH-302 induced γH2AX and apoptosis were also increased upon the addition of Chk1 inhibitor. Potentiation of TH-302 cytotoxicity by Chk1 inhibitor was only observed in cell lines proficient in, but not deficient in homology-directed DNA repair. We also show that combination treatment led to lowering of Rad51 expression levels as compared to either agent alone. In vivo data demonstrate that Chk1 inhibitor enhances TH-302 anti-tumor activity in p53 mutant HT-29 human tumor xenografts, supporting the hypothesis that these in vitro results can translate to enhanced in vivo efficacy of the combination.ConclusionsTH-302-mediated in vitro and in vivo anti-tumor activities were greatly enhanced by the addition of Chk1 inhibitors. The preclinical data presented in this study support a new approach for the treatment of p53-deficient hypoxic cancers by combining Chk1 inhibitors with the hypoxia-activated prodrug TH-302.

Abstract A147: Pixantrone induces cell death through mitotic perturbations and subsequent aberrant cell divisions in solid tumor cell lines.

Abstract Background and Aim: Pixantrone (Pix) is an aza-anthracenedione which has demonstrable activity in patients with non-Hodgkin's lymphoma. Pix has both alkylating and intercalating activity, but the mechanism of cell killing is unclear. Here we sought to elucidate the mechanisms by examining the effects of Pix on a number of cancer cells. Specifically, we assessed the impact Pix has on the cell cycle, DNA damage response and their relationships to cell killing. Methods: Cell lines derived from ovarian, colorectal, pancreatic and breast cancers were tested for response to Pix. We used a combination of FACs and microscopy to determine whether Pix altered cell cycle kinetics. To determine effects on cell death, short term (MTS) and long term (clonogenic) assays were performed. Apoptosis was determined by Annexin V staining and by biochemical markers as ascertained by western blotting. Moreover, live cell video-microscopy was used to assesses mechanisms of cell death. Results: We found that concentrations of Pix that were sufficient to induce cell death in clonogenic assays did not perturb cell cycle dynamics. We show that sensitivity to Pix did not correlate with levels of topoisomerase II protein, arguing that cell death was not due to inhibition of topoisomerase II. Immunofluorescence staining showed that cells treated with killing doses of Pix lacked detectable phospho-H2AX foci. This contrasted with doxorubicin which induced robust phospho-H2AX foci that was accompanied by a G2 cell cycle arrest. However, we did find a number of DNA damage and repair proteins formed discrete foci after treatment including pATM, pChk1, and 53BP1. How this relates to the standard DNA damage response is currently unclear. In addition, treatment of cells with Pix interfered with chromosome segregation during mitosis that were detected by the presence of chromosome bridges during mitosis and formation of micro/multi-nuclei after cell division. Long-term studies showed that cells do not immediately die after aberrant division, but only die after 3-4 abnormal rounds of mitoses, consistent with our observations that cell viability was reduced only after 3-4 days of Pix treatment. We addressed whether p53 was a determinant of Pix sensitivity by testing a pair of isogenic cell lines which differed in p53 status. We found that cells lacking p53 were less sensitive to Pix and took longer to die (>3 days) as compared to cells with wildtype p53 cells. Given that pChk1 foci were detected in Pix-treated cells, we tested the effects of Chk1 inhibitors in combination with Pix. We found that the combination of treatments killed more efficiently than either drug alone. Conclusion: Our studies show that Pix does not elicit a DNA damage response that leads to cell cycle arrest. The mechanism of cell killing appears to be by impairing chromosome segregation that generates severely aneuploid cells. Interestingly, the aberrant progeny cells do not die immediately but continued to divide for approximately 4 divisions before dying. The molecular mechanisms by which Pix impairs mitosis remains to be elucidated. We propose that the generation of micro nuclei as a result of aberrant division may serve as a biomarker of Pix efficacy and is a concept we are testing further. Finally, the finding that Pix appears to synergize with Chk1 inhibitors opens a window of opportunity to enhancement of tumor cell killing. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A147. Citation Format: Neil Beeharry, Xijun Zhu, Vignish Murali, Mitchell R. Smith, Timothy Yen. Pixantrone induces cell death through mitotic perturbations and subsequent aberrant cell divisions in solid tumor cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A147.

The Efficacy of CHK1 Inhibitors Is Not Altered by Hypoxia, but Is Enhanced after Reoxygenation

Inhibitors of CHK1 are in clinical trials for cancer treatment in combination with DNA-damaging agents. Importantly, it was previously suggested that hypoxic cancer cells may be particularly sensitive to CHK1 inhibition. However, this suggestion was based on studies in severe, toxic levels of hypoxia (anoxia). The influence of less severe hypoxia on the efficacy of CHK1 inhibitors, administered either as single agents or in combination with other treatments, remains to be investigated. Here, we have assayed the effects of the CHK1 inhibitors, AZD7762 and UCN-01, during various hypoxic conditions and after reoxygenation in the absence and presence of ionizing radiation. Treatment with CHK1 inhibitors during acute or prolonged hypoxia (< 0.03%, 0.2%, and 1% O2; 3 h or 20-24 h) gave similar effects on cell survival as treatment with these inhibitors during normoxia. CHK1 inhibitors combined with ionizing radiation showed similar radiosensitization in hypoxic and normoxic cells. However, when the inhibitors were administered after reoxygenation following prolonged hypoxia (< 0.03% and 0.2%; 20-24 h), we observed decreased cell survival and stronger induction of the DNA damage marker, γH2AX, in S-phase cells. This was accompanied by enhanced phosphorylation of the single-stranded DNA-binding replication protein A. These results suggest that the cytotoxic effects of CHK1 inhibitors are enhanced after reoxygenation following prolonged hypoxia, most likely due to the increased replication-associated DNA damage. Combining CHK1 inhibitors with other treatments that cause increased reoxygenation, such as fractionated radiotherapy, might therefore be beneficial.

Tumour selective targeting of cell cycle kinases for cancer treatment

The deregulation of the cell cycle and checkpoint machinery in cancer presents a highly attractive therapeutic strategy. We review here the strategies used to exploit the dysregulated cell cycle, both through targeting kinases required for cell cycle progression, and checkpoint kinases to inappropriately force cells through the cell cycle. Appropriate control of the cell cycle is critical for proliferating normal cells, and we discuss the importance of defining tumour specific vulnerabilities that could be targeted with cell cycle kinase inhibitors. Recent studies have shown that ER-positive breast cancers rely on CDK4 to promote proliferation. TP53 mutant cancer cell lines are sensitive to WEE1 and CHK1 inhibitors in combination with chemotherapy, while PTEN-deficient aneuploid cancer cell lines are sensitive to TTK inhibitors.

Abstract 3289: Mechanistic studies of the potentiation of hypoxia-targeted drug TH-302 by co-administration of checkpoint kinase1 (Chk1) inhibitors.

Abstract The hypoxia-activated prodrug TH-302 is reduced at its nitroimidazole group and under hypoxic conditions, releases Br-IPM, a bis-alkylating DNA crosslinker. We previously reported that Chk1 inhibitors are TH-302 sensitizers both in vitro and in vivo in the context of p53 checkpoint deficiency. To assess the impact of the Chk1 inhibitor AZD7762 on DNA damage in TH-302 treated cells directly, we used the single-cell electrophoresis-based ‘Comet’ assay. Cells treated with vehicle, TH-302, or AZD7762 exhibited no visible tail moment. In contrast, cells treated with both AZD7762 and TH-302 exhibited a tail moment, indicating the induction of double-strand breaks (DSBs). To determine the effect of AZD7762 on TH-302-induced DNA damage response, γH2AX was evaluated. We demonstrated in p53−/− HT29 cells that either TH-302 or AZD7762 induced γH2AX. However, treatment with both AZD7762 and TH-302 greatly increased γH2AX staining, demonstrating a greater DNA damage response with the combiniaton therapy. Induction of apoptosis was also assessed. TH-302 did not induce apoptosis in HT29, while AZD7762 caused a slight increase in apoptosis. However, treatment with both TH-302 and AZD7762 induced a dramatic increase in apoptosis, indicating that the Chk1 inhibitor AZD7762 sensitizes HT29 cells to apoptosis in response to the DNA damage induced by TH-302. To explore involved signaling pathways, we conducted immunoblot analysis and observed an increase in phospho-histone H3 at serine 10 in the combination treatment group, consistent with DNA damaged cells undergoing mitotic catastrophe. To investigate which DNA repair system is affected by Chk-1 inhibitor-mediated enhancement of TH-302 cytotoxicity, we utilized CHO cell-based DNA repair mutant cell line pairs. Potentiation of TH-302 cytotoxicity by AZD7762 was only observed in cell lines proficient in, and not deficient in, the specific type of DNA repair involved in the repair of TH-302 DNA damage (homology-dependent repair; HDR). To explore the role of HDR in AZD7762-mediated potentiation of TH-302 cytotoxicity further, we investigated the effects of AZD7762 on Rad51 levels. In response to TH-302, Rad51 expression levels were increased. However, combination treatment of cells with AZD7762 and TH-302 led to Rad51 expression levels lower than those observed in control cells. The results suggest that the down-regulation of Rad51-dependent HDR by AZD7762 results in the persistence of unrepaired DNA damage in response to TH-302 and underlies the mechanism of potentiation of TH-302 cytotoxicity by AZD7762. The preclinical data presented in this study support a new approach for the treatment of cancer by combining Chk1 inhibitors with the tumor-hypoxia targeted TH-302. Citation Format: Fanying Meng, Deepthi Bhupathi, Charles P. Hart. Mechanistic studies of the potentiation of hypoxia-targeted drug TH-302 by co-administration of checkpoint kinase1 (Chk1) inhibitors. [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 3289. doi:10.1158/1538-7445.AM2013-3289

Abstract 1784: Enhancement of hypoxia-activated prodrug TH-302 activity by Chk1 inhibition

Abstract TH-302 is a hypoxia-activated prodrug currently in clinical trials for the treatment of cancer. TH-302 releases the DNA cross-linking bromo-isophosphoramide mustard under hypoxic conditions. When DNA damage occurs, a signal transduction cascade is activated in response to the damage and transmits signals to the downstream effectors that connect with the cell cycle machinery. Checkpoint kinase 1 (Chk1) is a vital link between the upstream sensors of the DNA damage checkpoints and the cell cycle effectors. Generally, cell cycle progression is interrupted at the stage where the cell was when injured to give the cell time to repair the damage by activating DNA damage response and repair pathways. Due to its integral role in maintaining genomic integrity, Chk1 has been considered an attractive molecular target for inhibition to treat cancer. In recent years, Chk1 inhibitors have been studied for use in combination with DNA damaging anticancer agents that cause S and G2/M arrest in attempts to increase the efficacy of cancer treatment while sparing normal cells. We have shown that TH-302 induces G2/M arrest at low concentrations and a pan-cell cycle arrest at high concentrations. We hypothesized that the pharmacological inhibition of Chk1 kinase activity could potentiate the efficacy of TH-302. To investigate this possibility, we tested Chk1 inhibitors PF477736, AZD7762 and LY2603618 in combination with TH-302 in HeLa cervical, HT-29 colon, and H460 NSCLC cell lines. Employing an in vitro proliferation assay, we show that TH-302 activity is greatly enhanced (15 to 50 fold) by the addition of the Chk1 inhibitors in the two p53-deficient HeLa and HT29. In contrast, TH-302 activity is not affected by the presence of the Chk1 inhibitors in the p53 wild-type H460. The differential effects on TH-302 activity in combination with the Chk1 inhibitors were confirmed using clonogenic survival assays. These results are consistent with published studies showing p53 status playing a critical role in the activity of Chk1 inhibitors in combination with other DNA damaging agents. We hypothesized that TH-302 induced cell-cycle arrest at the G2/M phase may be mediated by activation of Chk1 and prevention of the activation of downstream Cdc2 kinase activity. We show that Chk1 inhibitors can abrogate TH-302-induced G2 checkpoint arrest in HeLa cells. Since Chk1 affects Cdc2 phosphorylation, we also evaluated the status of Cdc2 phosphorylation in response to the Chk1 inhibitor alone, TH-302 alone, or the combination of Chk1 inhibitor and TH-302. The results demonstrate that TH-302 alone, but not Chk1 inhibitor alone, increases Cdc2-Y15 phosphorylation due to the induced G2/M arrest. In the combination study, the addition of the Chk1 inhibitor blocks the TH-302-induced increase of Cdc-Y15 phosphorylation. Taken together, the results suggest a novel approach for the treatment of cancer combining Chk1 inhibitors with the tumor-hypoxia targeted TH-302. 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 1784. doi:1538-7445.AM2012-1784

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Role of checkpoint kinase 1 (Chk1) in the mechanisms of resistance to histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACi) are a new group of anticancer drugs with tumor selective toxicity. Normal cells are relatively resistant to HDACi-induced cell death compared with cancer cells. Previously, we found that vorinostat induces DNA breaks in normal and transformed cells, which normal but not cancer cells can repair. In this study, we found that checkpoint kinase 1 (Chk1), a component of the G2 DNA damage checkpoint, is important in the resistance of normal cells to HDACi in vitro and in vivo. Inhibition of Chk1 activity with Chk1 inhibitor (UCN-01, AZD7762, or CHIR-124) in normal cells increases their sensitivity to HDACi (vorinostat, romidepsin, or entinostat) induced cell death, associated with extensive mitotic disruption. Mitotic abnormalities included loss of sister chromatid cohesion and chromosomal disruption. Inhibition of Chk1 did increase HDACi-induced cell death of transformed cells. Thus, Chk1 is an important factor in the resistance of normal cells, and some transformed cells, to HDACi-induced cell death. Use of Chk1 inhibitors in combination with anticancer agents to treat cancers may be associated with substantial toxicity.

Abstract 2549: Efficacy of the Chk1 inhibitor PF-00477736 and pemetrexed in human mesothelioma

Abstract Malignant pleural mesothelioma (MPM) is a highly aggressive malignancy with the median survival of less than 1 year from diagnosis. Pemetrexed (PTX), a multi-targeted antifolate drug in combination with cisplatin, has been used as front-line treatment. PTX-treatments causes S-phase cell cycle arrest allowing cancer cells to repair damaged DNA critical for cell survival and proliferation. The Chk1 kinase, a central regulator of cell cycle progression and a principal mediator of cell cycle checkpoints in response to genotoxic stress is overexpressed in mesothelioma. We evaluated the in-vitro activity of the Chk1 kinase inhibitor, PF-00477736, in combination with PTX in MPM. PF-00477736 treatments abrogated the PTX-induced S-phase cell cycle arrest and enhanced cytotoxicity of PTX in a dose-dependent manner (∼81% cell proliferation inhibition, vs. ∼13% with PTX alone). PTX and PF-00477736 treatments alone enhanced the levels of γH2AX (6% and 16%, respectively), however, their combination had significantly higher effect (∼60% increase) compared to control cells. These results suggest that when Chk1 is inhibited, PTX-induced replication stress is converted into additional DNA damage due to checkpoint abrogation and lack of DNA repair. The sensitization of mesothelioma cells to PTX by PF-00477736 was also associated with a marked increase in apoptosis (∼11-fold increase compared to PTX of PF-00477736 treatments alone). Collectively, these results show a therapeutic potential of combining Chk1 inhibitors and PTX for the treatment of mesothelioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2549. doi:10.1158/1538-7445.AM2011-2549

P53‐dependent G1 arrest in 1st or 2nd cell cycle may protect human cancer cells from cell death after treatment with ionizing radiation and Chk1 inhibitors

This study was performed to explore the strategy of combining Chk1 inhibitors with ionizing radiation (IR) to selectively target p53-deficient cancer cells. Survival and cell cycle progression were measured in response to IR and the Chk1 inhibitors, UCN-01 and CEP-3891, in colon carcinoma HCT116 p53+/+ and p53-/- cells, and in osteosarcoma U2OS-VP16 cells with conditional expression of dominant-negative p53 (p53DD). Clonogenic survival was selectively reduced in HCT116 p53-/- compared to p53+/+ cells after treatment with UCN-01 and IR, and HCT116 p53+/+ cells also displayed strong p53-dependent G(1) arrest in the 1st cell cycle after IR. In contrast, clonogenic survival was affected similarly in U2OS-VP16 cells with and without expression of p53DD. However, death of U2OS-VP16 cells was p53 dependent as assessed by cell viability assay at 72 h, and this was associated with p53-dependent G(1) arrest in the 2nd cell cycle after treatment. Notably, HCT116 cells were overall more resistant than U2OS cells to cytotoxic effects of Chk1 inhibitors. Our results suggest that p53-dependent G(1) arrest in both 1st and 2nd cell cycles may protect human cancer cells from cell death after treatment with IR and Chk1 inhibitors. However, a challenge for future clinical use will be that different cancers display different intrinsic sensitivity to such inhibitors.

Abstract A13: Decitabine treatment of c-Myc transformed cancer cells is augmented by the removal of the checkpoint kinase Chk1

Abstract Decitabine, or DacogenTM (also known as 5-Aza-2′-deoxycytidine), is a FDA approved drug currently in clinical use against myelodysplastic syndrome, a pre-stage of acute myeloid leukemia (AML). Decitabine is a DNA methyltransferase inhibitor which causes rapid genome-wide demethylation, reactivating genes silenced during tumorigenesis. We have recently shown that Decitabine is a valid therapeutic agent in treatment of c-Myc transformed tumor cells and that overexpression of c-Myc sensitized mouse embryonic fibroblasts to Decitabine treatment. In vivo modeling using cells overexpressing c-Myc showed a delayed tumor development in animals treated with a single dose of Decitabine. Futhermore, Decitabine treatment was shown to cause DNA damage that was further relayed in a p53-independent manner through the checkpoint kinase Chk1. In the light of this, we decided to combine Decitabine treatment with a commercially available Chk1 inhibitor (SB218078). In this study we show that combinatorial treatment with Chk1 inhibitor and Decitabine augments the separate treatment regimes. Furthermore, we also show that RNAi against Chk1 sensitizes tumor cells from the λ-Myc transgenic mice to Decitabine treatment. This demonstrates the therapeutic possibilities of using Chk1 inhibitors in combination with DNA damage agents in treatment of c-Myc transformed cells. Citation Information: Cancer Res 2009;69(23 Suppl):A13.