Despite the enthusiasm surrounding cancer immunotherapy, most SCLC patients show very modest response to immune checkpoint inhibitor monotherapy treatment. Therefore, there is growing interest in ...combining immune checkpoint blockade with chemotherapy and other treatments to enhance immune checkpoint blockade efficacy. Based on favorable clinical trial results, chemotherapy and immunotherapy combinations have been recently approved by the U.S. Food and Drug Administration for frontline treatment for SCLC.
Here, we show that combined treatment of SRA737, an oral CHK1 inhibitor, and anti–programmed death ligand 1 (PD-L1) leads to an antitumor response in multiple cancer models, including SCLC. We further show that combining low, non-cytotoxic doses of gemcitabine with SRA737 + anti–PD-L1/anti–PD-1 significantly increased antitumorigenic CD8+ cytotoxic T cells, dendritic cells, and M1 macrophage populations in an SCLC model. This regimen also led to a significant decrease in immunosuppressive M2 macrophage and myeloid-derived suppressor cell populations, as well as an increase in the expression of the type I interferon beta 1 gene, IFNβ, and chemokines, CCL5 and CXCL10.
Given that anti–PD-L1/anti–PD-1 drugs have recently been approved as monotherapy and in combination with chemotherapy for the treatment of SCLC, and that the SRA737 + low dose gemcitabine regimen is currently in clinical trials for SCLC and other malignancies, our preclinical data provide a strong rational for combining this regimen with inhibitors of the PD-L1/PD-1 pathway.
Objective The genetic basis of inflammatory bowel disease (IBD) is incompletely understood. The aim of this study was to identify rare genetic variants involved in the pathogenesis of IBD. Design ...Exome sequencing and immunological profiling were performed in a patient with early onset Crohn's disease (CD). The coding region of the gene encoding X-linked inhibitor of apoptosis protein (XIAP) was sequenced in samples of 275 paediatric IBD and 1047 adult-onset CD patients. XIAP genotyping was performed in samples of 2680 IBD patients and 2864 healthy controls. Functional effects of the variants identified were investigated in primary cells and cultured cell lines. Results Our results demonstrate the frequent occurrence of private variants in XIAP in about four percent of male patients with paediatric-onset CD. While XIAP mutations are known to be associated with the primary immunodeficiency (PID) X-linked lymphoproliferative disease type 2 (XLP2), CD patients described here exhibited intestinal inflammation in the absence of XLP2 and harboured a spectrum of mutations partially distinct from that observed in XLP2. The majority of XIAP variants identified was associated with a selective defect in NOD1/2 signalling, impaired NOD1/2-mediated activation of NF-κB, and altered NF-κB-dependent cytokine production. Conclusions This study reveals the unanticipated, frequent occurrence of XIAP variants in male paediatric-onset CD. The link between XIAP and NOD1/2, and the association of XIAP variants with XLP2, support the concept of PID in a subset of IBD patients. Moreover, these studies provide a rationale for the implementation of XIAP sequencing in clinical diagnostics in male patients with severe CD.
Abstract
DNA replication is a tightly regulated process required for faithful duplication of the genome. Previous research in lower eukaryotes has demonstrated a requisite role for the CDC7 protein ...kinase in DNA replication origin firing through phosphorylation of the MCM2-7 helicase complex. Owing to its overexpression in various neoplasms, CDC7 has emerged as an attractive target for cancer treatment. We previously reported that SRA141, a potent and selective CDC7 inhibitor, is cytotoxic to multiple tumor cell lines in vitro and demonstrates robust anti-tumor efficacy in colorectal and leukemia xenograft models. Here, we explored the effects of SRA141 on DNA replication and cell cycle dynamics and how these effects relate to the mechanism of SRA141-induced cell death in colorectal cancer cell lines.
SRA141 treatment caused a complete inhibition of MCM2 phosphorylation and reduced overall cellular DNA synthesis within 3 hours, with the greatest effects observed in late S-phase. Unexpectedly, DNA combing experiments indicated that SRA141 caused a 70-90% increase in the rate of replication fork progression. These findings suggest that SRA141 may indeed limit origin firing, but this effect is compensated for in part by increased replication fork rates. Despite delay, nucleotide analog pulse-chase experiments indicated that SRA141 treated cells ultimately finished S phase and transitioned into mitosis. In contrast to other reports, no evidence of replication fork collapse was observed, indicating that the main mechanism of SRA141 cytotoxicity is not mediated through replication fork collapse. Intriguingly, some of the most pronounced effects of SRA141 treatment were on progression through M phase, as evidenced by an accumulation of mitotic cells and increased Cyclin B levels following treatment. Moreover, inhibition of Aurora B kinase, which regulates mitotic progression, strongly synergized with SRA141 in caspase-dependent cancer cell killing. Taken together, our findings indicate that the mechanism of cytotoxicity of CDC7 inhibitors is distinct from agents that cause replication fork collapse or inhibit CDKs, and thus may define a new class of cancer therapeutic agents that synergize with drugs that target certain mitotic pathways.
Citation Format: Veena Jagannathan, Snezana Milutinovic, Ryan Hansen, Bryan Strouse, Christian Hassig, Eric Brown. CDC7 kinase inhibition by SRA141 induces a potentially novel caspase-dependent tumor cell apoptosis associated with altered DNA replication and cell cycle dynamics abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-288.
Abstract
Targeting the DNA damage response (DDR) network is a promising strategy for the development of new cancer therapies. Checkpoint kinase 1, Chk1, is a central mediator of the DDR network and ...the potent, selective oral Chk1 inhibitor, SRA737, is being investigated in clinical trials. A distinct class of DDR inhibitors targeting PARP (PARPi) are approved for the treatment of ovarian cancers; however, tumors with functional homologous recombination (HR) repair are less sensitive to their effects, thereby limiting the clinical potential of these agents. Several reports have described the synergistic combination of Chk1i and PARPi, although the mechanism of anti-tumor activity has not been well defined. We explored the efficacy and mechanism of cytotoxicity of SRA737 in combination with the PARPi, niraparib, in HR repair proficient tumor cell lines. In short-term cell viability assays, the combination of SRA737 and niraparib elicited greater tumor cell death than either agent alone, as early as 12 hours after exposure to drug. Combination indices determined from colony forming assays indicated synergistic activity (CI < 0.7) using clinically achievable concentrations of each agent. Quantitative immunofluorescence studies revealed activation of ATM and phosphorylation of H2AX within 4 hours of treatment, indicating induction of DNA double strand breaks and activation of DDR signaling. Concurrent changes in the phosphorylation of mTOR, AMPK and the downstream target ULK1 suggested an induction of autophagy. Consistent with this hypothesis, the single agents, as well as the combination, led to decreases in p62 and LAMP2 levels and simultaneous increases in ATG5 and Beclin1 expression, and ATG13 phosphorylation. Autophagic flux was confirmed in cells expressing an LC3-GFP-RFP reporter plasmid. Genetic knockdown of autophagy components resulted in partial rescue of cell viability, suggesting that autophagy-dependent cell death may represent a mechanism of cytotoxicity for this DDR combination. Given that rescue of cell death was incomplete following abrogation of autophagy, we additionally examined the involvement of apoptotic pathways. SRA737 and niraparib treatment resulted in reduced levels of anti-apoptotic proteins, BCL-XL and MCL-1, and increased levels of the pro-apoptotic protein, BIM. Moreover, knock down of pro-apoptotic proteins or over-expression of anti-apoptotic proteins partially rescued combination-induced lethality. Collectively these results argue that toxic autophagy, as well as the intrinsic and extrinsic apoptosis pathways, contribute to SRA737 and niraparib-induced tumor cell killing. The involvement of multiple mechanisms of cell death may decrease the likelihood of cancer cells to acquire resistance to these agents. These findings support further investigation of SRA737 in combination with PARPi, including niraparib, in HR repair proficient cancers.
Citation Format: Laurence Booth, Jane Roberts, Andrew Poklepovic, Ryan J. Hansen, Bryan Strouse, Snezana Milutinovic, Christian Hassig, Paul Dent. The Chk1 inhibitor, SRA737, synergizes with niraparib to kill cancer cells via multiple cell death pathways 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 1853.
DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of ...DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122–417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.
Abstract
Background: Small cell lung cancer (SCLC), the most aggressive form of lung cancer, shows poor response rates to immunotherapy targeting the programmed cell death protein 1 pathway ...(PD-(L)1). Our group previously discovered that SCLC exhibits high expression of checkpoint kinase 1 (CHK1) and that the CHK1 inhibitor SRA737 activates the innate immune STING pathway, demonstrating robust anti-tumor activity and synergy in combination with anti-PD-L1 in an SCLC model. As SRA737 is being tested in SCLC patients in combination with low dose gemcitabine (LDG), we evaluated the efficacy and immune correlates (including macrophages associated with resistance to immune checkpoint blockade) of the SRA737+LDG regimen in combination with anti-PD-L1 in an SCLC model.
Results: Trp53, Rb1 and p130 (RPP) triple knockout SCLC cells were implanted into the flank of B6129F1 immunocompetent mice. After the mice developed tumors, they were treated with single agents or various drug combinations. Anti-PD-L1 and LDG demonstrated minimal effect on tumor growth as single agents and only a modest effect as a combination. Moderate to strong anti-tumor activity was however observed with SRA737 monotherapy which directly correlated with dosing intensity. The most profound and synergistic anti-tumor activity was observed when anti-PD-L1 was combined with the SRA737+LDG regimen, with all animals showing durable regressions. Analysis of tumor infiltrating immune cells at the end of this treatment regimen showed a dramatic induction of cytotoxic T-cells and a reduction of exhausted and regulatory T cells. Similarly, pro-inflammatory M1 type macrophages and dendritic cells were increased while immunosuppressive M2 type macrophages and MDSC cells were dramatically decreased. As monotherapy, the more dose intensive SRA737 schedule resulted in similar effects on lymphocytes when combined with anti-PD-L1. These effects are consistent with our previous data showing that SRA737 treatment leads to an induction of STING and type I interferon signaling in tumors, which is associated with the establishment of an anti-tumor immune microenvironment.
Discussion: Our findings suggest that the combination of anti-PD-L1 with the SRA737+LDG regimen may represent the optimal implementation of these agents, leading to a dramatic anti-tumor activity accompanied by the establishment of a strong anti-tumor immune microenvironment. Given that anti-PD-(L)1 drugs are approved but show limited efficacy in SCLC, our preclinical data provide a strong rationale for combining these agents with the SRA737+LDG regimen to enhance clinical response rates.
Citation Format: Triparna Sen, Carminia M. Della Corte, Snezana Milutinovic, Lixia Diao, Robert J. Cardnell, Ryan J. Hansen, Bryan Strouse, Michael P. Hedrick, Christian Hassig, Jing Wang, Lauren A. Byers. Combination treatment of the CHK1 inhibitor, SRA737, and low dose gemcitabine demonstrates profound synergy with anti-PDL1 inducing durable tumor regressions and modulating the immune microenvironment in small cell lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-148.
Abstract
High grade serous ovarian cancers (HGSOC) have defective homologous recombination (HR) genes in 50% of cases, while a distinct 20% demonstrate CCNE1 amplification (CCNE1amp). HR-deficient ...HGSOC are initially sensitive to Poly(ADP-ribose) polymerase inhibitors (PARPi) but drug resistance ultimately emerges. CCNE1amp HGSOC show resistance to PARPi and platinum treatments. Here, we investigated the anti-tumor activity of the potent, highly selective, orally bioavailable small molecule inhibitor of Checkpoint kinase 1 (Chk1), SRA737, in both acquired PARPi-resistant and CCNE1amp HGSOC models. HR-deficiencies and CCNE1amp are known to increase replication stress, leading to increased reliance on Chk1, a key regulator of cell cycle progression and the replication stress response. We hypothesized that Chk1 inhibition by SRA737 will result in increased replication stress, inducing subsequent cell death and tumor regression in both PARPi-resistant and CCNE1amp ovarian cancer models. In colony formation assays, SRA737 monotherapy decreased cell survival in HR-deficient, PARPi-resistant and CCNE1amp cells. Additionally, the combination of SRA737 with PARPi was synergistic in decreasing colony formation in HR-deficient (PEO1, best coefficient of drug interaction (CDI)=0.53; JHOS4, CDI=0.45) and PARPi-resistant cell models (PEO1-PR, CDI=0.11; PEO4, CDI=0.08). SRA737 treatment led to a dose-dependent increase in the replication stress marker pCHK1 (S345), confirming an on-target drug effect in PARPi-resistant (PEO1-PR) and CCNE1amp (OVCAR3) cells. Furthermore, treatment with SRA737 induced gH2AX (indicator of DNA damage) which increased modestly in combination with PARPi. SRA737 was also evaluated in a PARPi-resistant PDX model as well as in CCNE1amp in vivo mouse models. Preliminary evidence in a PARPi resistant PDX model demonstrated tumor growth inhibitory activity of SRA737 in combination with PARPi. Consistent with in vitro activity, SRA737 inhibited tumor growth in an OVCAR3 xenograft model. Lastly, in an orthotopic PDX model established from a platinum-resistant CCNE1amp ovarian cancer patient, SRA737 monotherapy caused significant tumor regression, similar to SRA737 in combination with PARPi. Strategies to optimize treatments for PARPi-resistant HGSOC, as well as for platinum-resistant CCNE1amp HGSOC, are needed. In PARPi-resistant models, SRA737 is active as a monotherapy, and the combination of SRA737 with PARPi demonstrated synergy. In CCNE1amp tumors, SRA737 showed profound activity as a monotherapy in this PARPi-resistant model. SRA737 is a new potent and selective Chk1 inhibitor that demonstrated activity in acquired PARPi-resistant as well as CCNE1amp preclinical cancer models, warranting further development in these HGSOC subgroups.
Citation Format: Haineng Xu, Sergey Medvedev, Ashka Pandya, Hyoung Kim, Yasuto Kinose, Eric Brown, Ryan J. Hansen, Bryan Strouse, Snezana Milutinovic, Christian Hassig, Fiona Simpkins. The novel oral Chk1 inhibitor, SRA737, is active in both PARP inhibitor resistant and CCNE1 amplified high grade serous ovarian cancers 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 LB-265.
The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess ...mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2′-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novosynthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such asNF-κB, JunB, ATF-3, and GADD45β (growth arrestDNA damage 45β gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information.
Abstract
Replication stress (RS) is a primary source of genomic instability, tumorigenesis, and cancer progression. RS is defined as an uncoupling of the replicative helicase and DNA polymerase, ...resulting in long stretches of fragile single stranded DNA (ssDNA) that is prone to damage. Excessive RS can result in replication catastrophe and cell death, which could be leveraged as a therapeutic strategy to treat high-RS cancers. While overexpression of certain oncogenes has been implicated as a driver of RS in cancer, the precise cellular conditions that result in RS have been difficult to predict. Here we demonstrate that high copy number amplification of genes on non-native extrachromosomal DNA (ecDNA) is associated with elevated RS in comparison to the same amplification on linear chromosomes. The ecDNA bearing cells display heightened basal levels of phosphorylated RPA, a hallmark of ssDNA, and decreased replication fork velocities. Consistent with this finding, the ecDNA amplified cells display greater sensitivity to inhibitors of deoxyribonucleotide production relative to cells harboring chromosomal gene amplification and to normal cells. Moreover, ecDNA bearing cells demonstrate a dramatic vulnerability to reduction in the conditionally essential amino acid glutamine, the primary nitrogen source for de novo nucleotide biosynthesis. The enhanced sensitivity to glutamine starvation is directly correlated with a reduction in ecDNA in tumor cells, and a corresponding selection for chromosomally integrated gene amplification in the surviving population. These phenotypes are rescued with nucleoside supplementation but not anaplerotic TCA cycle intermediates, further reinforcing a link between ecDNA and RS biology. Collectively, observations made here support a novel mechanism for oncogene-induced RS shaped by ecDNA driven oncogene amplification, revealing a novel molecular strategy to identify high-RS tumors and a therapeutic approach to target this “Achilles heel” of tumor biology.
Citation Format: Sudhir Chowdhry, Salvador Garcia, Nam-Phuong Nguyen, Anthony Celeste, Edison Tse, Snezana Milutinovic, Deepti Wilkinson, Christian Hassig, Shailaja Kasibhatla. Replication stress and the inability to repair damaged DNA, the potential “Achilles' heel” of ecDNA+ tumor cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1520.
Abstract
Background: Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer. Despite the recent success of immunotherapy in other indications, only a minority of SCLC patients ...respond to immune checkpoint blockade (ICB) targeting programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) either as a monotherapy or combination. Therefore, there is a strong need to develop strategies to enhance the efficacy of immunotherapy in SCLC. Our group previously discovered that SCLC exhibits high expression of checkpoint kinase 1 (Chk1) and showed that preclinical in vivo models of SCLC respond to Chk1 inhibition. Based on data from others and our group, we hypothesized that targeting Chk1 can enhance antitumor immunity and synergize with ICB.
Results: SRA737 treatment decreased cell viability with a range of potencies in a panel of SCLC cell lines in vitro. This was accompanied by an induction of double-strand breaks in sensitive cell lines as demonstrated by increased γ-H2AX. Intriguingly, SRA737 also led to an increase in micronuclei formation and STING activation in cells in vitro. Cell surface and total PD-L1 protein were increased following SRA737 treatment in vitro, further supporting a potential benefit of combining the drug with immune checkpoint blockade therapy in vivo. As hypothesized, SRA737 showed strong synergy with anti-PD-L1 antibody in an immunocompetent xenograft SCLC model. Triple-knockout SCLC cells generated from a GEMM mouse model with conditional deletion of Trp53, Rb1 and p130 were implanted into the flank of B6129F1 mice. The mice were treated for three weeks with either IgG (control), SRA737 (100mg/kg, either 3/7 or 5/7 days), anti-PD-L1 (300ug, 1/7 days) or the combination. While anti-PD1 antibody treatment was largely ineffective, SRA737 significantly delayed tumor growth (at Day 21: T/C=0.30 for 3/7 days and T/C=0.28 for 5/7 days). Combination treatment with SRA737 and anti-PD-L1 demonstrated remarkable antitumor efficacy, resulting in stable disease following SRA737 schedule of 3/7 days (T/C=0.12) and tumor regressions following SRA737 schedule of 5/7 days (T/C=0.1). These effects were sustained after treatment cessation and the long-term survival benefit is being assessed.
Discussion: The intrinsic antitumor activity of the Chk1 inhibitor, SRA737, was significantly enhanced by addition of an anti-PD-L1 antibody, leading to tumor regressions in an immunocompetent SCLC model. Preliminary evidence suggests SRA737 induces micronuclei formation, STING activation and PD-L1 expression in tumor cells. Further studies to elucidate the mechanism of Chk1 inhibition-induced antitumor immunity in SCLC are ongoing. SRA737 is currently being tested in clinical trials both as a monotherapy and in combination with other agents. Given that the anti-PD-L1 antibody opdivo is now approved for SCLC, our data suggest intriguing possibilities for therapeutic synergy between the oral Chk1 inhibitor, SRA737, and ICB therapy that warrant further clinical investigation.
Citation Format: Triparna Sen, Snezana Milutinovic, Robert J. Cardnell, Lixia Diao, Youhong Fan, Ryan J. Hansen, Bryan Strouse, Michael P. Hedrick, Christian Hassig, Jing Wang, Lauren A. Byers. The oral Chk1 inhibitor, SRA737, synergizes with immune checkpoint blockade in small-cell lung cancer (SCLC) abstract. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B15.
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