Kaufmann, Scott H.

Primary Myelofibrosis (PMF) is an aggressive myeloproliferative neoplasm (MPN) characterized by constitutional symptoms, cytopenias, splenomegaly, extramedullary hematopoiesis, bone marrow (BM) fibrosis, and a propensity to transform to acute myeloid leukemia (AML). Despite advances in understanding the underlying genetic abnormalities in MPN and the development of JAK2 inhibitors to treat MF, there is an urgent need to devise new treatment strategies, particularly for triple negative myelofibrosis (TN-MF) cases that lack mutations in the JAK2 kinase pathway and who have inferior outcomes. To identify potential oncogenic drivers in TN-MF, we performed cytogenetic analyses and targeted exome sequencing of 98 genes commonly mutated in myeloid malignancies in 584 MF patients identified from the Total Cancer Care database at Moffitt Cancer Center. Notably, there were no significant differences in the somatic mutation profiles (other than JAK2 activating mutations) between TN vs. JAK2/CALR/MPL mutant MF patients. However, trisomy of chromosome 8 occurs more frequently in TN-MF vs. other subtypes (26.7% vs. 6.2%, p=0.0001). To identify oncogenic drivers in trisomy 8+ TN-MF, we performed scRNA-seq analysis from 18,651 cells ( n=15,850 from 3 normal donors and n=2,801 from a trisomy 8+ TN-MF patient) and identified a total of 1,260 genes that are differentially regulated in hematopoietic stem cells (HSCs) of trisomy 8+ TN-MF compared to normal donors. Among these, 131 genes were located on chromosome 8 and MYC was one of the top 5 genes that are significantly upregulated in trisomy 8+ HSCs. Additional immunohistochemistry staining demonstrated that MYC protein levels in BM cells were significantly higher in trisomy 8+ TN-MF patients vs. trisomy 8- patients. To assess whether MYC might be a driver of MPN, we established an Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC transgenic mouse model that inducibly overexpresses MYC in HSCs following treatment of polyinosinic-polycytidylic acid (pIpC). Forced MYC expression induced profound anemia, monocytosis, megakaryocytic atypia, splenomegaly, increased BM collagen deposition/fibrosis, extramedullary hematopoiesis in spleen and liver, and significantly reduced OS vs. Mx1-Cre +/- wild type controls (median OS 258 days vs. not reached (NR), p<0.0001). MYC also promoted expansion of HSCs, myeloid progenitors, and Gr-1 +/CD11b + mature myeloid cells in BM and spleen; thus, MYC was sufficient to provoke MF-like disease independent of JAK2 pathway mutations. Subsequent scRNA-seq analysis of 25,232 cells ( n=13,552 from control and n=11,680 from a MYC transgenic mouse) revealed that S100a9 mRNA levels were increased by MYC in most major hematopoietic cell types. Supporting oncogenic roles of S100A9 in MYC-driven MF, deletion of S100a9 in Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC;S100a9 -/- mice abolished MYC-induced MF phenotypes and significantly improved OS (median OS NR vs. 225 days, p=0.0345). To test whether inhibition of MYC can suppress MF disease progression in vivo, lethally irradiated CD45.1 +/CD45.2 + WT mice were transplanted with BM cells harvested from CD45.2 + Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC mice that were treated with pIpC 20 weeks prior to transplant. MYCi975, a small molecule that inhibits MYC, reduced MYC and S100a9 protein levels in BM and spleen cells. Further, MYCi975 effectively suppressed MYC-driven MF phenotypes and disease progression, and significantly improved OS (median OS NR vs. 252 days, p=0.0109). In summary, our studies are the first to describe an oncogenic role of MYC in MF pathogenesis, where MYC provokes an alarmin-driven inflammatory circuit, and where the MYC-S100A9 axis represents a therapeutic vulnerability for TN-MF patients. Accordingly, our results provide a strong rationale for testing agents targeting MYC or S100A9 in early phase clinical trials in MPNs having increased MYC levels or activity.

AbstractBackgroundDespite recent approval of several new agents, relapsed acute lymphoblastic leukemia (ALL) remains challenging to treat. Sapanisertib (MLN0128/TAK‐228) is an oral TORC1/2 inhibitor that exhibited preclinical activity against ALL.MethodsWe conducted a single‐arm multi‐center Phase II study of sapanisertib monotherapy (3 mg orally daily of the milled formulation for 21 days every 28 days) in patients with ALL through the Experimental Therapeutics Clinical Trials Network (NCI‐9775).ResultsSixteen patients, 15 of whom were previously treated (median 3 prior lines of therapy), were enrolled. Major grade 3–4 non‐hematologic toxicities included mucositis (3 patients) and hyperglycemia (2 patients) as well as hepatic failure, seizures, confusion, pneumonitis, and anorexia (1 patient each). Grade >2 hematological toxicity included leukopenia (3), lymphopenia (2), thrombocytopenia, and neutropenia (1). The best response was stable disease in 2 patients (12.5%), while only 3 patients (19%) were able to proceed to Cycle 2. Pharmacokinetic analysis demonstrated drug exposures similar to those observed in solid tumor patients. Immunoblotting in serially collected samples indicated limited impact of treatment on phosphorylation of mTOR pathway substrates such as 4EBP1, S6, and AKT.ConclusionIn summary, single‐agent sapanisertib had a good safety profile but limited target inhibition or efficacy in ALL as a single agent. This trial was registered at ClinicalTrials.gov as NCT02484430.

Abstract Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of genome integrity in mammalian cells. In this study, we report a new role for the Krüppel-type ZNF-containing protein ZNF432 as a novel poly(ADP-ribose) (PAR) reader that regulates the DNA damage response. We show that ZNF432 is recruited to DNA lesions via DNA- and PAR-dependent mechanisms. Remarkably, ZNF432 stimulates PARP-1 activity in vitro and in cellulo. Knockdown of ZNF432 inhibits phospho-DNA-PKcs and increases RAD51 foci formation following irradiation. Moreover, purified ZNF432 preferentially binds single-stranded DNA and impairs EXO1-mediated DNA resection. Consequently, the loss of ZNF432 in a cellular system leads to resistance to PARP inhibitors while its overexpression results in sensitivity. Taken together, our results support the emerging concept that ZNF-containing proteins can modulate PARylation, which can be embodied by the pivotal role of ZNF432 to finely balance the outcome of PARPi response by regulating homologous recombination.

Abstract Background: Ovarian cancer ranks fifth in cancer deaths among women. The gold standard of care for advanced disease includes cytoreductive surgery along with adjuvant or neoadjuvant platinum/taxane-based chemotherapy. Although response to this treatment is high, chemotherapy-induced nausea and vomiting due to platinum agents and allergic reactions due to both agents are common. For nearly half a century, the glucocorticoid (GC) dexamethasone (DEX) has been administered as part of supportive care. However, there have periodically been questions about the possible deleterious effects of DEX on chemotherapy response, especially in solid tumors. Methods: Platinum-sensitive, glucocorticoid receptor (GRα) expressing ovarian cancer cells were exposed to DEX prior to chemotherapy and clonogenic survival was assessed. The effects of DEX on cell cycle and apoptosis were assessed by flow cytometry. Changes in the tumor cell transcriptome after acute DEX treatment were determined by RNA sequencing. Mice bearing orthotopic ovarian cancer patient-derived xenografts (PDXs) were treated with chemotherapy ± DEX to assess changes in chemotherapy response in vivo. Findings: DEX pre-treatment decreased platinum cytotoxicity in ovarian cancer cell lines in vitro and diminished platinum response in some PDXs analyzed for chemotherapy response ex vivo. RNA-seq analysis revealed changes in transcripts encoding proteins involved in platinum detoxification and TNFα pro-survival signaling. In ovarian cancer PDXs, DEX decreased platinum response in one model and had limited impact on both platinum and platinum/taxane chemotherapy in three distinct studies. Interpretation: DEX appears to modulate chemotherapy response in some settings in vitro and ex-vivo. The impact of DEX on chemotherapy response in vivo appears to be limited. However, further investigation of this question in vivo in non-immunosuppressed murine models appears warranted. Citation Format: Annapoorna Venkatachalam, Cristina Correia, Cordelia D. Mcgehee, Sarah Finstuen-Magro, Jamison VanBlaricom, Paula A. Schneider, Kevin L. Peterson, Ethan P. Heinzen, Melissa C. Larson, Xiaonon Hou, Ann L. Oberg, Saravut John Weroha, Scott H. Kaufmann. Impact of dexamethasone on chemotherapy response in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5058.

Abstract Ovarian cancer is one of the most common gynecologic malignancies, with high-grade serous carcinoma (HGSC) as the most frequent and deadliest type of ovarian cancer (OC). With the recent advances in cancer epigenomic profiling, it has become clear that intra-tumor heterogeneity driven by genetic and epigenetic factors may be the basis of resistance, where current treatments could efficiently target sub-populations of cells while a sub-group may be resistant, or prone to develop resistance. With epigenomic profiling and single cell sequencing technology, we aim to characterize cell signatures that translate as prognostic markers and novel drug targets. We have mapped the epigenomic landscape of HGSC in 12 patient-derived xenografts (PDX), and their metastatic counterparts (circulating tumor cells and ascites present in the PDX models) using an assay for transposase-accessible chromatin (or ATAC-seq) and compared it with normal fallopian tube organoids to identify tumor and metastatic-specific regulatory elements. We also performed RNA-seq to validate potential transcriptional dependencies associated with resistance to chemotherapy. In order to identify potentially chemotherapy-resistant subpopulations and further validate the activity of some of the transcription factors that can be driving resistance, we performed single cell multiomic profiling using the 10x Multiome sequencing platform (scRNA+scATAC from the same nuclei) in HGSC patient samples and PDX. Finally, we performed immunohistochemistry of patient tissue slides for target markers and correlated it with overall survival in OC patients. As previously suggested, a subset of cancer clonal cells can drive treatment resistance and metastasis and result in poor patient outcomes. Data derived from PDX ATAC-seq showed a cancer stem cell signature driven by SOX2 in metastatic samples compared to the paired PDX primary tumor, suggesting a role for SOX2 in disease progression. Interestingly, SOX2 expression in epithelial cells in the Multiome analysis is more patient-specific than other stem cell markers, like CD133. We also observed that SOX2 expression negatively correlated with prognosis. These data, taken together, point out the promising role of SOX2 as a prognostic and targetable marker for HGSC. Citation Format: Leticia Assad Maia Sandoval, Wazim Mohammed Ismail, Amelia Mazzone, Amik Munankarmy, Jagneet Kaur, Tsering Tashi, Xiaonan Hou, Nagarajan Kannan, Ann Oberg, Scott H. Kaufmann, Saravut John Weroha, Alexandre Gaspar-Maia. Single cell multiomic profiling of high-grade serous carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4724.

Abstract Ovarian cancer is the 7th most common cancer in women and the most fatal of all female reproductive cancers, with 5-year overall survival of 10-30%. Despite initial responses to first line platinum- and paclitaxel-based chemotherapies, >70% of ovarian cancers recur with increasingly resistant disease; and nearly all of these women die of their disease. Recently, the use of targeted therapies such as bevacizumab and PARP inhibitors have been shown to improve progression-free survival in a subset of patients. However, these agents have thus far failed to improve overall survival in patients without specific genetic signatures, highlighting the continued need for alternative therapies. Through an unbiased drug screen, we have identified lestaurtinib as a potent inhibitor of many sensitive and resistant ovarian cancer cell lines and patient derived organoid models. Lestaurtinib is a known tyrosine kinase inhibitor originally developed to block FLT3, a protein that is not expressed in any of the ovarian cancer cell lines tested. To elucidate the mechanisms by which lestaurtinib inhibits ovarian cancer cells, we have examined its impact on the phospho-proteome in parallel with RNAseq. Results of these studied have identified inhibition of the JAK/STAT signaling pathway as a top hit. Assessment of STAT1 and STAT3 has revealed that these transcription factors are constitutively activated in chemotherapy- and PARP inhibitor-resistant cell lines as reflected by high levels of Tyr701/705 and Ser727 phosphorylation, suggesting that induction of this pathway may drive resistance. Surprisingly, profound differences in the ability of various JAK/STAT inhibitors to block these specific phosphorylation events is observed, which correlates with their potency. Further, STAT1 and STAT3 ChIPseq experiments following cytokine-mediated activation of this pathway reveal that these transcription factors bind to genomic regions encoding genes involved in oncogenic and drug resistance pathways. Genetic knockdown of STAT1 and STAT3 via siRNA, or knockout via CRISPR/Cas9, results in significant growth inhibition of sensitive and resistant models of ovarian cancer, confirming their importance in maintaining cell viability. Finally, combining lestuartinib with standard-of-care cisplatin or olaparib (a PARP inhibitor) is shown to be synergistic, indicating that pharmacological inhibition of JAK/STAT signaling has the potential to counteract drug resistance. Ongoing studies are aimed at further understanding the role of JAK/STAT signaling in ovarian cancer, elucidating the mechanistic processes by which STAT1/3 mediate progression of this disease, and identifying the most effective pharmacological strategies to study in possible future clinical trials. Citation Format: Esther Rodman, Michael Emch, Elizabeth Bruinsma, Xiaonan Hou, Scott Kaufmann, Saravut J. Weroha, John Hawse. Investigation of the JAK/STAT signaling pathway in chemotherapy and PARP inhibitor resistant ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4862.

AbstractBackgroundCyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo‐ovarian high‐grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large‐scale, histotype‐specific validation has been performed. The hypothesis was that high‐level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC.MethodsWithin the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated.ResultsHigh‐level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high‐level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08‐1.47, p = .034, and HR, 1.18; 95% CI, 1.05‐1.32, p = .015, respectively). This was also true for cases with combined high‐level amplification/overexpression (HR, 1.26; 95% CI, 1.09‐1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1‐SD increase; 95% CI, 0.94‐1.06; p = .58). CCNE1 high‐level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss.ConclusionThis study provides large‐scale validation that CCNE1 high‐level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.

Mutations in homologous recombination (HR) genes, including BRCA1 , BRCA2 , and the RAD51 paralog RAD51C , predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.

Abstract Background: The kinases ataxia-telangiectasia-mutated-and-Rad3-related (ATR), checkpoint kinase 1 (CHK1) and WEE1 participate in the response to replication stress. Inhibitors of ATR, WEE1 and CHK1 are being tested for antineoplastic activity as monotherapies and in combination with chemotherapy. Although these agents impair cell cycle checkpoints, fork stabilization, origin firing and homologous recombination (HR), little is known about the apoptotic pathways that are engaged and the critical mediators activating cell death mechanisms. The present study utilizes high grade serous ovarian cancer (HGSOC) cell lines and patient derived xenografts (PDXs) to identify the primary mechanism of cell death in ovarian cancer and assess changes involved in the resistance setting after treatment with these inhibitors. Methods: A panel of HGSOC cell lines was examined for responses to ceralasertib (ATRi), prexasertib (CHK1i) or adavosertib (WEE1i) using colony forming assays, immunoblotting and assays for apoptosis. Mechanisms involved in acquired resistance and downstream changes in cell fate were evaluated by generating prexasertib-resistant HGSOC cell lines. Subsequent NGS analysis was performed in the sensitive and resistant pairs. Results: Irrespective of the homologous recombination (HR) status, ovarian cell lines and PDXs underwent cell death during prolonged exposure at clinically achievable concentrations. Treatment of ovarian cancer cell lines with ATRi, CHK1i or WEE1i activated the replication checkpoint, consistent with previously published reports. The subsequent apoptotic response involved PMAIP1 and BCL2L11 upregulation rather than the TNFa-induced death receptor-mediated apoptosis we recently described in acute leukemia (Cancer Res. PMID: 33414171). Additionally, ovarian lines selected for CHK1i resistance failed to die in response to either ATRi or WEE1i. Conclusions: Prexasertib, ceralasertib, and adavosertib exhibit monotherapy activity in both HR deficient and proficient HGSOC cell lines and PDX models. Unlike AML, death receptor mediated apoptosis was not observed; instead, the primary mechanism of cell killing in HGSOC lines involves activation of the mitochondrial apoptotic pathway. Cross-resistance of these DNA damage repair modulators is likely multifactorial and mainly involves diminished replication stress response. Citation Format: Annapoorna Venkatachalam, Kevin L. Peterson, Cristina Correia, Karen S. Flatten, Xianon Hou, Paula A. Schneider, Emily Balczewski, Cordelia McGehee, Rachel M. Hurley, Xue W. Meng, Chance Sine, Rameen Shah, Nicole Vincelettte, Husheng Ding, Hu Li, Saravut (John) Weroha, Scott H. Kaufmann. CHK1 inhibitor prexasertib induces NOXA-dependent apoptosis in ovarian cancer [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 1832.