Schneider, Paula A.

Abstract Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.

Abstract PARP inhibitor (PARPi) resistance in high-grade serous ovarian carcinoma (HGSOC) can be acquired as a result of restored homologous recombination (HR) due to secondary or reversion mutations in HR genes, such as BRCA1, BRCA2, and RAD51C, or due to loss of BRCA1 promoter methylation (meBRCA1). We have demonstrated that homozygous meBRCA1 can be lost or reverted to heterozygous methylation following treatment with platinum-based chemotherapy, resulting in HR-competent PARPi-resistant tumors. RAD51C promoter methylation (meRAD51C) is detected in approximately 2% of HGSOC cases and, as with meBRCA1, is associated with gene silencing and HR deficiency. We are exploring PARPi response in meRAD51C preclinical models to determine clinical relevance. Two patient-derived xenograft (PDX) models of HGSOC with RAD51C gene silencing caused by meRAD51C have distinct meRAD51C profiles (measured by methylation-specific high-resolution melt analysis and targeted bisulfite next-generation sequencing), and different responses to PARPi treatment pressure. PDX PH039 loses methylation and regains RAD51C expression after only 2 cycles of PARPi retreatment (niraparib), resulting in PARPi-refractory tumors by cycle 3-4. Illumina EPIC methylation array analysis of PH039 revealed increasing global methylation losses following each round of PARPi treatment. Lack of meRAD51C stability and rapid development of PARPi resistance in PH039 may be due to the high degree of meRAD51C heterogeneity within the tumor favoring selection of pre-existing HR-competent clones under PARPi pressure. In contrast, PDX 183 has a relatively homogeneous and stable meRAD51C profile. We have multiple examples of using unique PDX models to demonstrate various important features of PARPi resistance, including loss of promoter methylation for BRCA1 vs. RAD51C. Thus, meRAD51C confers response to PARPi in HGSOC, but PARPi treatment pressure can cause loss of methylation and drug resistance in some tumors. The contrasting PARPi responses of these PDX provide a platform for the study of meRAD51C stability in vivo and may present therapeutic opportunities to improve meRAD51C durability and PARPi responses in patients. Citation Format: Kasenija Nesic, Rachel M Hurley, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Giada V. Zapparoli, Ashan Musafer, Ming E. Wong, John Weroha, Xiaonan Hou, Hu Li, Vivian Negron, Kevin Peterson, Paula Schneider, Elizabeth M. Swisher, Melissa Southey, Alexander Dobrovic, Matthew Wakefield, Scott H. Kaufmann, Clare L. Scott. DNA repair gene promoter methylation patterns adapt and influence PARP inhibitor response [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA02.

Abstract Introduction: Topoisomerase I is a nuclear enzyme that relaxes torsional strain in DNA by nicking one DNA strand, allowing rotation, then resealing the DNA backbone. This is an important enzyme in replication and transcription, and therefore has been targeted by anti-cancer agents such as camptothecins. These agents intercalate into DNA at the topoisomerase I active site and inhibit the religation step catalyzed by the enzyme, causing increased topoisomerase I-DNA complexes that impede advancing replication forks or transcription complexes, leading to DNA damage and cell death. Platinating agents also stabilize topoisomerase I-DNA covalent complexes. The topotecan/carboplatin combination has recently shown promise in combination with the PARP inhibitor veliparib in aggressive and transformed myeloproliferative neoplasms (MPNs) and chronic myelomonocytic leukemia (K Pratz et al., Clin Cancer Res. 23:899, 2017). Given that both classes of agents have a narrow therapeutic window, it is important to try to predict the sensitivity/resistance of these agents in malignancies such as acute myeloid leukemia (AML) and transformed MPNs. Some of mechanisms of drug resistance that have been described are decreased numbers of drug-stabilized topoisomerase I-DNA complexes due to drug efflux, diminished topoisomerase I expression or topoisomerase I mutation. Thus, being able to detect and quantify these DNA-protein complexes may provide insight into drug sensitivity. Methods: Using a unique monoclonal antibody that detects topoisomerase I-DNA covalent complexes (A. Patel et al., Nucleic Acids Res. 44:2816, 2016), immunofluorescence techniques were used to quantify these topoisomerase I-DNA complexes in AML cell lines (SET2, HEL, K562, U937, ML2, MV-4-11, THP1, HL60, ML1, and Molm13) and clinical AML samples. To verify the fidelity of the anti-topoisomerase I-DNA covalent complex antibody (TopIcc), siRNA knockdown methods were utilized with confirmatory immunofluorescence and Western blotting. Slot blots were additionally used to assess the presence of these complexes in AML cell lines. Western blots were performed to assess topoisomerase I levels in AML cell lines. Topotecan drug sensitivity analysis was performed by using an MTS assay. Results: With treatment of the topoisomerase I inhibitor, topotecan, there was an increase in the percentage of leukemia cells positive for the topoisomerase I-DNA covalent complexes. The degree of increase in complexes varied across cell lines and patient samples. To ensure we were detecting TopIcc by immunofluorescence, we performed a topoisomerase-I siRNA knockdown to confirm the specificity of the antibody for TopIcc's. Slot blots on multiple AML cell lines confirmed the presence of topoisomerase-DNA covalent complexes. The Western blot of all the AML cell lines, ordered from low to high TopIcc revealed increasing amounts of topoisomerase I. The MTS assay revealed lower topotecan IC50 values in three of the cell lines with higher complex formation, including Molm13, ML1 and ML2 (IC50 6-8 nM compared to 13-46 nM in the other cell lines). Conclusions: Based on recent clinical results, there has been renewed interested in topoisomerase I inhibitors, especially for aggressive and transformed MPNs. Topoisomerase I-DNA complex repair/downregulation has been a hallmark of drug resistance to topoisomerase I inhibitors. Therefore, detecting and quantifying these complexes in treated samples has been hypothesized to provide insight into the drug sensitivity, which is particularly important in camptothecins and platinating agents given their narrow therapeutic windows. Immunodetection techniques using an anti-TopIcc antibody may help predict sensitivity of AML to topoisomerase I inhibitors and will be used as a correlative biomarker study in an upcoming phase II randomized study of topotecan/carboplatin with or without veliparib or placebo in advanced myeloproliferative disorders and chronic myelomonocytic leukemia (ClinicalTrials.gov NCT03289910). Disclosures Pratz: AbbVie: Consultancy, Research Funding; Agios: Research Funding; Astellas: Consultancy, Research Funding; Boston Scientific: Consultancy; Millenium/Takeda: Research Funding. Kaufmann:Mayo Clinic: Patents & Royalties: Co-inventor on a patent held by Mayo Clinic for the use of the anti-TOP1cc antibody as a diagnostic reagent.

Abstract Abstract 970 The mammalian target of rapamycin, mTOR, is a highly conserved serine/threonine kinase known to play a role in regulating mRNA translation, cell cycle progression, cell proliferation and apoptosis. As a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, mTOR is a component of two distinct complexes, TORC1 and TORC2. While TORC1 facilitates cell cycle progression from G1 into S phase by phosphorylating p70S6 kinase and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), TORC2 catalyzes the activating phosphorylation of Akt on Ser473, providing a feedback loop for further activation of mTOR. Phase II trials have shown activity of the TORC1-selective inhibitor rapamycin and its analogs in a wide range of lymphoma subtypes. The purpose of this study was to evaluate the anti-proliferative and pro-apoptotic effects of the dual TORC1/TORC2 inhibitor OSI-027 in human neoplastic lymphoid cells in vitro. MTS assays demonstrated that OSI-027 inhibited proliferation in a wide range of lymphoid lines, including SeAx (Sezary syndrome), DoHH2 (large cell lymphoma), RL (follicular lymphoma) and Jurkat (T cell ALL), as well as clinical lymphoma and T cell ALL samples, with IC50 values ranging from 0.078 to 10 μM. Propidium iodide staining followed by flow cytometry for subdiploid cells revealed induction of apoptosis within 48 h of treatment with OSI-027 (but not rapamycin) in SeAx, DoHH2, and Jurkat cells. Examination of Jurkat variants with alterations in key proteins involved in the death receptor versus mitochondrial pathway revealed diminished apoptotic responses to OSI-027 when Bcl-2 was overexpressed or caspase 9 was silenced, indicating involvement of the mitochondrial pathway. Immunoblotting for Bcl-2 family members revealed upregulation of Bim and Puma after a 48-hour exposure to OSI-027 but not rapamycin. This upregulation was also seen at the mRNA level, with a 12- to 20-fold increase in Puma mRNA and 4- to 12-fold induction of Bim mRNA. Small interfering RNA (siRNA)-mediated knockdown of Bim and Puma significantly diminished the apoptotic response to OSI-027. Because the Foxo3a transcription factor has been implicated in Bim and Puma expression and is known to be activated when Akt is inhibited, we next examined whether Bim and Puma induction was Foxo3a-dependent. Luciferase reporter assays showed that OSI-027 activated the full-length Puma and Bim promoters and that this activation was diminished when the Foxo3a binding sites were deleted or mutated. In addition, OSI-027 induced nuclear translocation of Foxo3a, while Foxo3a siRNA diminished OSI-027-induced apoptosis in Jurkat cells. Collectively, these results indicate that OSI-027 inhibits proliferation and induces apoptosis in a wide range of neoplastic lymphoid cells through a process that involves Foxo3a-mediated upregulation of Bim and Puma. These results also suggest that dual inhibition of TORC1 and TORC2 may be an effective treatment strategy in lymphoid malignancy. Disclosures: Barr: OSI Pharmaceuticals: Employment. Witzig:Novartis and Celgene: Patents & Royalties, Research Funding, Served on advisory boards with Novartis and Celgene – both uncompensated with compensation to Mayo Clinic.

Abstract Abstract 287 Purpose: Farnesyltransferases are key cellular enzymes involved in the prenylation of proteins. Prenylated proteins are important for malignant cell growth. The purpose of this phase II study (NCT00082888) was to assess tumor response and toxicity associated with the oral farnesyltransferase inhibitor tipifarnib (R115777, Johnson and Johnson) in patients with relapsed aggressive, indolent, or uncommon lymphoma. The uncommon group included low incidence lymphoma types such as Hodgkin lymphoma (HL) and T-cell non-Hodgkin lymphoma- cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). Patients and Methods: Patients with relapsed or refractory non-Hodgkin (NHL) or Hodgkin (HL) were eligible if they had measurable disease, performance status ≤2, platelet count ≥75,000 × 10(6)/L, an absolute neutrophil count ≥1000 × 10(6)/L and a creatinine ≤2 × upper limit of normal. Patients initially received tipifarnib 300 mg twice daily, days 1–21 every 28 days. The trial was conducted by the University of Iowa/Mayo Clinic Lymphoma SPORE. Results: Ninety-three patients with lymphoma (42 aggressive, 15 indolent, and 36 uncommon) enrolled in the trial between March 2004 and November 2008. The median age was 62 years (range, 18–91). The median number of prior therapies was 5 (range, 1–17). The overall response rate (ORR) was 20% (19/93) with 6% (6/93) complete responses (CR) and 14% (13/93) partial responses (PR). The ORR was 17%, 7%, and 31% for the aggressive, indolent, and uncommon groups, respectively (Table). Among the 19 responders were 7 diffuse large B-cell lymphoma (DLBCL), 7 T-cell NHL, 1 follicular grade II, and 4 HL. The overall median response duration for the 19 responders was 7.5 months (mean, 12.8; range, 1.8 – 48.1 months). The median response duration was 11.3 months (mean, 10.1; range, 1.8 – 18.5 months), 2 months, and 7.5 months (mean, 15.5; range, 1.9 – 48.1 months) for the aggressive (7), indolent (1), and uncommon (11) groups, respectively. Five patients in the uncommon group are still receiving treatment with a range of treatment duration between 25 – 60+ months. Among the 5 still receiving treatment 4 responded and 1 is stable. The range of response duration is 15.4, 17.7, 43.1, and 48.1 months. These five patients (2 with HL, 1 anaplastic large cell cutaneous, and 2 PTCL) are currently receiving doses of 400 (1), 200 (2), and 100 (2) mg twice daily. The median time to progression for all patients was 3.6 months (95% CI: 2.1 – 4.5 months). The grade 3,4 toxicities observed were primarily fatigue (10%) and reversible myelosuppression with 11% anemia, 37% neutropenia, and 32% thrombocytopenia. Correlative studies demonstrated tipifarnib-induced upregulation of Bim, a marker of tipifarnib action in lymphoma cell lines, in 7 of 9 paired lymphoma biopsies. Conclusions: Tipifarnib has activity in lymphoma, with responses in patients with heavily pretreated DLBCL, HL and T-cell types, but little activity in follicular NHL. This agent has an excellent toxicity profile and targets a novel pathway. Further studies in combination with other agents are warranted for patients with DLBCL, HL or T-cell NHL. Disclosures: No relevant conflicts of interest to declare.

Abstract The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in hematologic malignancies. Rapamycin analogues, which partially inhibit mTOR complex 1 (mTORC1), showed limited anti-tumor activity due to feedback mechanisms involving mTORC2 and incomplete inhibition of mTORC1. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR kinase domain. The purpose of this study was to delineate the mechanisms of mTOR dual inhibitor induced apoptosis in human neoplastic lymphoid cells in vitro. MTS assays and propidium iodide staining followed by flow cytometry for subdiploid populations demonstrated that OSI-027 and MLN0128 inhibited cell proliferation and induced apoptosis in a wide range of lymphoid cell lines including Jurkat, Nalm-6, Molt-4, and SeAX. Raptor and Rictor knockdown in Jurkat and Nalm-6 increased cell death, suggesting both mTOR complexes play a role in apoptosis. 4EBP1 phosphorylation was inhibited by mTOR dual inhibitors, but not by rapamycin. Expression of 4EBP1 T37A/T46A and 4EBP1 T37A/T46A/S65A/T70A, which mimic dephosphorylated 4EBP1, increased Puma mRNA and protein levels as well as apoptosis. Moreover, 4EBP1 knockdown abrogated mTOR dual inhibitor induced Puma upregulation and cell death, further supporting the role of 4EBP1 dephosphorylation in mTORC1 dependent apoptosis. In accord with the known dependence of c-Myc translation on the eIF4E/eIF4G complex, we also observed c-Myc downregulation after treatment with OSI-027, MLN0128 and 4EGI-1, but not rapamycin. Puma induction mirrored c-Myc downregulation under a variety of conditions, including expression of nonphosphorylatable 4EBP1 in parental Jurkat cells or wt 4EBP1 in 4EBP1 deficient cells. Furthermore, c-Myc knockdown induced Puma mRNA and protein as well as increased apoptosis. Collectively, these results support a model in which mTORC1 inhibition, acting through 4EBP1, induces Puma upregulation and apoptosis through c-Myc downregulation. In order to assess the parallel mTORC2-dependent Bim-mediated apoptotic mechanism, we utilized reporter assays and RNAseq experiments. OSI-027-induced Bim promoter activity decreased markedly when the nucleotides -29 to -18 were removed, suggesting that this response element is critical for OSI-027-induced promoter activation. In silico analysis identified eight transcription factors, including SP1, Egr-1, and Myb, that potentially bind this 12-bp region. In RNAseq experiments, we detected a 9-fold increase in Egr-1. Egr-1 upregulation was confirmed by qRT-PCR and immunoblotting after dual inhibitors treatment or Rictor knockdown. Moreover, dominant negative Egr-1 or Egr-1 knockdown diminished dual inhibitor-induced Bim promoter activation and Bim upregulation. Chromatin immunoprecipitation assays demonstrated that OSI-027 enhances binding of Egr-1 to a region of the Bim promoter including bp -29 to -18, further confirming that Egr-1 functions as a direct transcriptional activator for Bim upon mTOR dual inhibitor treatment. NFκB is a known transcription factor for Egr-1 and we observed increased p65 in the nucleus and increased NFκB transcriptional activity after dual inhibitor treatment. Overexpression of S32A/S36A IκB impaired the ability of dual inhibitors to induce NFκB transcriptional activation, Egr-1 mRNA and protein, Bim promoter activation and Bim mRNA and protein upregulation. Collectively, these results suggest that mTORC2 inhibition induces Bim upregulation and apoptosis through NFκB and Egr-1 transactivation. When fresh clinical ALL isolates were exposed to OSI-027 or MLN0128 ex vivo, inhibition of 4EBP1 phosphorylation along with upregulation of Egr-1 and Bim and/or c-Myc downregulation accompanied by Puma induction occurred, indicating that the pathways identified in ALL cell lines can also potentially be engaged in clinical ALLs. These observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Acquired PARP inhibitor (PARPi) resistance in high-grade serous ovarian cancer (HGSOC) as a result of restored homologous recombination has been observed following secondary mutations that restore full-length protein in BRCA1, BRCA2, RAD51C, and RAD51D. Additionally, loss of BRCA1 methylation has also been shown to confer resistance. However, little is known about the role of RAD51C methylation in acquired PARPi resistance. In ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib in ovarian carcinoma, four (2%) tumors demonstrated RAD51C methylation. The present study utilizes HGSOC patient derived xenografts (PDXs) and recurrent samples from ARIEL2 to assess the role of RAD51C methylation in the development of PARPi resistance. Methods: To drive PARPi resistance, PDX039, an extremely PARPi-sensitive model lacking demonstrable mutations in DNA repair genes, was treated cyclically with niraparib (100 mg/kg) for 21 days, after which the tumor was allowed to regrow and re-established in new mice for the next treatment round. To evaluate the frequency of methylation change, RAD51C methylation was analyzed in 12 rucaparib-treated mice (300 or 450 mg/kg) harboring PDX183, a PARPi-sensitive model without mutations in DNA repair genes. Global changes in gene expression following development of PARPi resistance were assessed by RNA sequencing. RAD51C promoter methylation was evaluated by bisulfite sequencing. Subsequent functional analysis included qRT-PCR, IHC, and western blot. DNA damage response pathways are being evaluated by immunofluorescence ex vivo following niraparib, rucaparib, or IR. Results: PDX039 grew through PARPi treatment by the third and fourth cycle of therapy. RAD51C was the only DNA repair gene to show significant change in RNAseq analysis (log2 fold-change=8.43; p=2e-192), corresponding with a loss of RAD51C methylation. Moreover, after just one round of PARPi treatment, RAD51C methylation was lost in 1 of 12 PARPi-treated PDX183 xenografts. RAD51C methylation loss ultimately resulted in restoration of expression, for which functional analysis is ongoing. Analysis of patient samples is currently underway. Conclusions: In HGSOC PDX models, RAD51C methylation affords PARPi sensitivity in the absence of DNA repair gene mutations. Treatment pressure with PARPi can reverse RAD51C methylation and restore RAD51C expression. Isolated changes in methylation of the RAD51C locus are sufficient to restore HR and convey PARPi resistance. Citation Format: Rachel M. Hurley, Ksenija Nesic, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Paula A. Schneider, Xiaonan Hou, Cristina Correia, Karen S. Flatten, Giada V. Zapparoli, Alexander Dobrovic, Kevin K. Lin, Thomas C. Harding, Andrea E. Wahner Hendrickson, Elizabeth M. Swisher, Matthew Wakefield, S. John Weroha, Clare L. Scott, Scott H. Kaufmann. Loss of RAD51C promoter hypermethylation confers PARP inhibitor resistance [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 5885.

Key Points Agents that inhibit both complexes containing the mammalian target of rapamycin are particularly toxic to acute lymphocytic leukemia cells. This killing reflects engagement of a 4EBP1/c-MYC/PUMA axis downstream of mTORC1 and an NF-κB/EGR1/BIM axis downstream of mTORC2.

Key Points BCL2 mutations in FL correlate with activation-induced cytidine deaminase expression and frequently alter the amino acid sequence of the protein. Mutations in the BCL2 coding sequence at diagnosis are associated with shortened time to transformation and earlier death due to lymphoma.

Abstract Bcl-2 is phosphorylated on Ser70 after treatment of cells with spindle poisons. On the basis of effects observed in cells overexpressing Bcl-2 S70E or S70A mutants, various studies have concluded that Ser70 phosphorylation either enhances or diminishes Bcl-2 function. In the present study, the ability of phosphorylated Bcl-2, as well as the S70E and S70A mutants, to bind and neutralize proapoptotic Bcl-2 family members under cell-free conditions and in intact cells was examined in an attempt to resolve this controversy. Surface plasmon resonance indicated that phosphorylated Bcl-2, Bcl-2 S70E, and Bcl-2 S70A exhibit enhanced binding to Bim and Bak compared with unmodified Bcl-2. This enhanced binding reflected a readily detectable conformation change in the loop domain of Bcl-2. Furthermore, Bcl-2 S70E and S70A bound more Bak and Bim than wild-type Bcl-2 in pull-downs and afforded greater protection against several chemotherapeutic agents. Importantly, binding of endogenous Bcl-2 to Bim also increased during mitosis, when Bcl-2 is endogenously phosphorylated, and disruption of this mitotic Bcl-2/Bim binding with navitoclax or ABT-199, like Bcl-2 downregulation, enhanced the cytotoxicity of paclitaxel. Collectively, these results provide not only a mechanistic basis for the enhanced antiapoptotic activity of phosphorylated Bcl-2, but also an explanation for the ability of BH3 mimetics to enhance taxane sensitivity. Cancer Res; 73(23); 6998–7008. ©2013 AACR.