The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination ...through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2(lex1)) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2(lex1) mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2(lex1) mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51. Copyright 2003 Wiley-Liss, Inc.
Abstract
HGSOC (characterized by defects in DNA damage repair and high levels of genomic instability and DNA replication stress) is the most lethal gynecological malignancy in the United States. The ...current treatment options for HGSOC are limited and new approaches are needed. Checkpoint kinase 1 (Chk1), a key protein kinase that regulates the cell cycle, DNA damage and replication stress response, has emerged as an attractive target for anti-cancer therapy. Prexasertib, an ATP-competitive inhibitor of Chk1, is being evaluated in a Phase 2 trial sponsored by NCI (NCT02203513); and the preliminary data showed encouraging results in patients with wild-type BRCA HGSOC. PI3K/AKT/mTOR pathway plays key roles in cancer cell survival, homologous recombination repair and drug resistance. Our previous data showed the expression level of genes related to PI3K/AKT signaling is elevated in prexasertib-resistant TNBC patient-derived xenograft (PDX) tumors. In this study we explored whether the PI3K/AKT/mTOR pathway is involved in prexasertib response of HGSOC and evaluated the combination effect of prexasertib with a PI3K/mTOR inhibitor (LY3023414) on HGSOC in vitro and in vivo. Data from 24 n=1 HGSOC PDX tumors indicated that the tumors with high phospho-AKT were resistant to prexasertib. The PI3K/AKT/mTOR signaling (including p70S6K and S6RP) was activated and correlated with the increased DNA damage (γH2AX) in HGSOC cell lines treated with prexasertib. These data suggest that the PI3K/AKT signaling is associated with prexasertib resistance and the cell survival response to DNA damage induced by prexasertib; and provide the scientific rational for a prexasertib/LY3023414 combination. Indeed the prexasertib/LY3023414 combination induced synergistic or additive inhibition on cell proliferation and colony formation in multiple HGSOC cell lines. This combination enhanced replication stress (phospho-RPA32), DNA damage (γH2AX), S phase arrest and cell death (cleaved PARP and sub-G1) in HGSOC Ovcar-8 cells when compared with single agent activity. Combination efficacy was further tested in HGSOC OV-90 and Cov504 xenograft models. Prexasertib induced tumor inhibition by 58.8% in OV-90 and tumor regression by 37.3% in Cov504, and LY3023414 induced tumor inhibition by 43.37% and 66% in OV-90 and Cov504 tumors, respectively. Prexasertib/LY3023414 combination resulted in tumor regression by 8.9% and 73.7% in OV-90 and Cov504 tumors, respectively, and significantly (p<0.001) enhanced efficacy when compared to singe agent. Taken together, these data support a potential combination strategy of Chk1 inhibitor prexasertib with a PI3K/mTOR inhibitor LY3023414 to treat HGSOC patients. The safety of this combination is being assessed in an ongoing Phase 1b clinical trial (NCT02124148).
Citation Format: Jack Dempsey, Greg Donoho, Philip Iversen, Jennifer Stephens, Ann McNulty, Ricardo Martinez, Christoph Reinhard, Louis Stancato, Aimee Lin, Wenjuan Wu. Inhibition of PI3K/AKT/mTOR signaling by a dual PI3K/mTOR inhibitor (LY3023414) potentiates the antitumor efficacy of the Chk1 inhibitor prexasertib (LY2606368) in models of human high-grade serous ovarian cancer (HGSOC) 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 1761.
Abstract
Reported genomic alterations in the DNA repair pathway in studies of castrate-resistant prostate cancer (CRPC) patients have prompted interests in targeted therapies related to the DNA ...damage response (DDR). CHK1 kinase plays a critical role in mediating the DNA damage checkpoint response by contributing to orderly cell cycle progression, regulation of the mitotic spindle-assembly checkpoint response and is essential for homologous recombination repair. The CHK1 kinase (CHK1) inhibitor prexasertib (LY2606368) is currently in clinical development. In this study we investigated the efficacy of prexasertib as single agent in CRPC in vitro cell lines and in vivo tumors. Prexasertib inhibited cell proliferation of prostate cancer cell lines showing the more consistent and potent inhibition in androgen-receptor positive (AR) + models (n=5, IC50 value ranges from 4.3 to 13.1 nM) while AR- cell lines had IC50 values ranging from 6.4 nM to 1000 nM (n=8). A sub-set of cell lines including VCAP, LNCaP, 22RV1 and PC3 underwent additional in vitro studies including cell cycle regulation and programmed cell death induced by prexasertib. A 24-hour treatment with 50 nM prexasertib increased S-phase populations in all cell lines (VCAP, LNCaP, 22RV1 and PC3) and sub-G1 populations in VCAP and 22RV1 cells. Live-cell imaging showed 50 nM prexasertib triggered caspase 3/7 induction by 30, 19, 15 and 10 fold change in VCAP, 22RV1, LNCaP and PC3, respectively when compared with control. Western blot studies characterized the activation of the DDR pathway signaling giving rise to a time- and concentration-dependent DNA damage response leading to induction of pCHK1 (S345), γH2AX, pRPA32(S4/8) and PARP cleavage. Importantly, in all three AR+ cell lines, prexasertib yielded both time- and concentration-dependent inhibition of AR-full length and AR-variant7 expression. In addition, prexasertib demonstrated similar single-agent activity in prostate cancer patient-derived organoid (PDO) models by inhibiting proliferation and increasing apoptosis. Finally, in vivo n=1 studies of six patient-derived xenograft (PDX) models which represent heavily pretreated mCRPC patients yielded single-agent prexasertib efficacy in 4/6 models. Similarly, xenograft models including 22RV1, LNCaP and PC3 also yielded single-agent efficacy compared to vehicle groups. On-going efforts continue to deepen the understanding of the mechanisms underlying the action of prexasertib and potential markers for drug response by using RNA/gene arrays. In conclusion, prexasertib yielded potent single-agent activity in preclinical studies including cancer cell lines, PDO, xenograft and PDX models of castrate-resistant prostate cancer and these data provide rationale of the development of the prexasertib for the treatment of CRPC.
Citation Format: Ann McNulty, Greg Donoho, Jack Dempsey, Adem Abel, Jennifer Stephens, Ricardo Martinez, Damien Gerald, Carole Perruzzi, Marguerita O’Mahony, Christoph Reinhard, Aimee Lin, Wenjuan Wu. The CHK1 kinase inhibitor prexasertib (LY2606368) shows potent single-agent efficacy in in vitro and in vivo models of castrate-resistant prostate 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 3513.
Abstract
Introduction: RAS mutations are present in 30% of non-small cell lung cancer (NSCLC) and, despite activated MAPK signaling, MEK inhibitors have disappointed in clinical trials. MAPK pathway ...reactivation and activation of anti-apoptotic signaling via the PI3K/mTOR axis are major resistance mechanisms, among others. ERK inhibitors are believed to overcome some of the restrictions of MEK inhibitors, and combinations with PI3K/mTOR inhibitors could potentially increase therapeutic efficacy. Past trials of MEK and PI3K inhibitor combinations, however, showed that toxicities and side effects are likely to be expected also for the ERK-PI3K/mTOR inhibitor combination. Therefore, more specific and potent drugs as well as alternating dosing schedules may overcome toxicities and improve treatment outcome.
Methods: We so far established five patient-derived RAS-mutant NSCLC cell lines (1/5 NRASQ61K, 1/5 KRASQ61K, 2/5 KRASG12C, 1/5 KRASG12D) and xenograft models (PDX), characterized them by next-generation sequencing (DFCI Oncopanel) and tested the antiproliferative (CellTiter-Glo® assay) and apoptosis-inducing (Caspase3/7 activity, IncuCyte® technology) efficacy of LY3214996 (ERK inhibitor) and LY3023414 (PI3K/mTOR inhibitor) in vitro. Effects on signal transduction were investigated by Western blot analysis.
Results: The ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 exhibit single-agent antiproliferative and apoptosis-inducing activity in patient-derived RAS-mutant NSCLC cell lines (for LY3214996: 1/5 sensitive (IC50<1μM), 2/5 intermediate sensitive (IC50<5μM), 2/5 resistant (IC50>10μM); for LY3023414: all IC50 <3μM, 3/5 <1μM). Despite reactivation of MAPK signaling (pMEK) upstream of ERK during LY3214996 treatment, ERK downstream targets (cMyc, DUSP4, SPRY2) were strongly suppressed. In sensitive cell lines, LY3214996 induced PARP cleavage and Bim accumulation in a dose- and time-dependent manner. Sensitivities to ERK and PI3K/mTOR inhibition varied between cell lines, indicating different pathway dependencies for proliferation and cancer cell survival.
Conclusions and Outlook: Both LY3214996 and LY3023414 have single-agent in vitro efficacy in patient-derived NSCLC models with different RAS mutations and co-mutational landscapes. Single-agent in vivo studies and tolerability studies of drug combinations are ongoing, and treatment predictors and intrinsic/adaptive resistance mechanisms will be investigated via RNAseq/GSEA and phospho-receptor tyrosine kinase arrays.
Citation Format: Jens Köhler, Prafulla C. Gokhale, Jiaqi Li, Margaret K. Wilkens, Hong Tiv, Shripad V. Bhagwat, Greg Donoho, Patrick Brueck, Ramon V. Tiu, Amanda J. Redig, Emily S. Chambers, Atsuko Ogino, Jihyun Choi, Man Xu, Paul Kirschmeier, Pasi A. Jänne. Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models 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 5467.
Abstract
Combination strategies leveraging chemotherapy and immunotherapy have held the promise as a method to improve benefit to cancer patients. However, most chemotherapies have detrimental ...effects on immune homeostasis and do not induce immunogenic cell death. The positive phase III trial of pemetrexed/carboplatin with the PD-1 antibody pembrolizumab in NSCLC (Keynote-189) lead to the first chemotherapy/immunotherapy combination ever approved. While this suggests a positive interaction between pemetrexed-based chemotherapy and PD-1 therapy, the underlying mechanism is unknown. Therefore, it is important to understand the role of pemetrexed in modulating antitumor immune response to assure application of this therapy to the appropriate patients. To characterize the effects of pemetrexed on intra-tumor immune response, murine tumor models which were sensitive to pemetrexed and known to be sensitive to PD-L1, were treated with pemetrexed with or without carboplatin, or anti-mouse PD-L1. In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intra-tumor leukocytes that was accompanied by immune-related gene expression changes indicative of enhanced T cell infiltration and/or activation. Gene expression induced by pemetrexed was largely unaffected by carboplatin. Treatment of both MC38 and Colon26 tumor cells in vitro with pemetrexed induced release of HMGB1, indicative of immunogenic cell death. Although proliferation of primary human T cells was slightly reduced by pemetrexed, at clinically relevant concentrations, treatment lead to an enhanced T cell activation phenotype, including upregulation of multiple interferon gamma-induced genes, and increased mitochondrial respiration. This correlated with improved antigen specific in vitro cytotoxic activity of OT-1 TCR transgenic CD8 T cells when treated with pemetrexed during priming with OVA peptide. Treatment with pemetrexed and PD-L1 demonstrated a combination benefit compared to either monotherapy in both tumor models. Pathway Analysis of gene expression data revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell-mediated immunity during the combination. This correlated with upregulation of MHC-I & II on monocytes, macrophages and tumor cells, suggesting increased immune priming. Accordingly, treatment with S1P1R antagonist (FTY720, preventing T cell LN egress) after initiation of therapy resulted in a loss of combination efficacy. This data suggests that pemetrexed promotes intra-tumor T cell-mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells. The combination of these effects results in enhanced T cell function leading to an improved anti-tumor efficacy in combination with PD-L1 antibody
Citation Format: David A. Schaer, Nelusha Amaladas, Zhao Hai Lu, Erik Rasmussen, Andreas Sonyi, Darin Chin, Andrew Capen, Yanxia Li, Catalina Meyer, Bonita Jones, Xiaodong Hong, Shuang Luo, Carmine Carpenito, Kenneth Roth, Alexander Nikolayev, Bo Tan, Manisha Brahmachary, Krishna Chodavarapu, Frank Charles Dorsey, Jason Manro, Thompson Doman, Greg Donoho, David Surguladze, Gerald Hall, Sandaruwan Geeganage, Michael Kalos, Ruslan Novosiadly. The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer immunotherapy via immunogenic tumor cell death and T cell-intrinsic mechanisms 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 3945.
Sequences of 13 lipoxygenases from various plant and mammalian species, thus far reported, display a motif of 38 amino acid residues which includes 5 conserved histidines and a 6th histidine about ...160 residues downstream. These residues occur at positions 494, 499, 504, 522, 531, and 690 in soybean lipoxygenase isozyme L-1. Since the participation of iron in the lipoxygenase reaction has been established and existing evidence based on Mossbauer and EXAFS spectroscopy suggests that histidines may be involved in iron binding, the effect of the above residues has been examined in soybean lipoxygenase L-1. Six singly mutated lipoxygenases have been produced in which each of the His residues has been replaced with glutamine. Two additional mutants have been constructed wherein the codons for His-494 and His-504 have been replaced by serine codons. All of the mutant lipoxygenases, which were obtained by expression in Escherichia coli, have mobilities identical to that of the wild-type enzyme on denaturing gel electrophoresis and respond to lipoxygenase antibodies. The mutated proteins H499Q, H504Q, H504S, and H690Q are virtually inactive, while H522Q has about 1% of the wild-type activity. H494Q, H494S, and H531Q are about 37%, 8%, and 20% as active as the wild type, respectively. His-517 is conserved in the several lipoxygenase isozymes but not in the animal isozymes. The mutant H517Q has about 33% of the wild-type activity. The inactive mutants, H499Q, H504Q, H504S, and H690Q, become insoluble when heated for 3 min at 65 degrees C, as does H522Q. The other mutants and the wild-type are stable under these conditions. Although the essentiality of His-499, -504, and -690 is not proven, they are tentatively considered to be prime candidates for iron ligands. Judgment on the role of H-522 is more uncertain, since mutant H522Q has weak but detectable activity. The Km values of the active mutants and the wild-type L-1, when determined against linoleic acid, differ only moderately, indicating that His replacements do not greatly influence the binding of the substrate
► High-content multiplexed methods to quantify immunohistochemistry. ► Quantification of tumor angiogenesis and vascular normalization. ► Quantification of tumor associated hypoxia, proliferation and ...apoptosis. ► Phenotypic characterization of multiple tumor models. ► Multiplexed tissue imaging to define mechanisms of actions of cancer therapeutics.
Targeting multiple hallmarks of cancer with drug combinations may provide unique opportunities for cancer therapeutics; however, phenotypic quantification is necessary to understand in vivo mechanisms of action of each drug alone or in combination. Immunohistochemistry (IHC) can quantify phenotypic changes, but traditional methods are not amenable for high-throughput drug discovery. In this article, we describe a high-content method to quantify changes in tumor angiogenesis, vascular normalization, hypoxia, tumor cell proliferation, and apoptosis using IHC. This method to quantify tumor model phenotypes can be useful for cancer drug discovery by increasing the understanding of: (i) tumor models used in efficacy studies, (ii) changes occurring during the growth of the tumor, and (iii) novel mechanisms of actions of cancer therapeutics.
Recent advances in automated imaging have enabled high-content quantification of tumor phenotypes to aid cancer drug discovery.
Patient-derived xenografts (PDX) have emerged as an important translational research tool for understanding tumor biology and enabling drug efficacy testing. They are established by transfer of ...patient tumor into immune compromised mice with the intent of using them as Avatars; operating under the assumption that they closely resemble patient tumors. In this study, we established 27 PDX from 100 resected gastric cancers and studied their fidelity in histological and molecular subtypes. We show that the established PDX preserved histology and molecular subtypes of parental tumors. However, in depth investigation of the entire cohort revealed that not all histological and molecular subtypes are established. Also, for the established PDX models, genetic changes are selected at early passages and rare subclones can emerge in PDX. This study highlights the importance of considering the molecular and evolutionary characteristics of PDX for a proper use of such models, particularly for Avatar trials.
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