Kaufmann, Scott H.

Abstract DAC combinations serve as frontline therapies for AML. DAC forms DNA-DNMT1 crosslinks and activates the ATR-CHK1-WEE1 DDR. Combinations of DNA-protein crosslinking agents with ATR, CHK1, or WEE1 inhibitors (i) have shown efficacy in solid tumors. This study evaluates the cytotoxic synergy of DAC plus ATRi, CHK1i, or WEE1i in AML. Public datasets were queried for mRNA expression and dependency. Commercial cell lines (U937, KG1a, Molm13, MV-4-11) were genomically validated. Molm13 TP53-/- clones were generated via CRISPR. Cells were cultured in complete RPMI. To treat, cells were cultured for 48 h in RPMI with drugs or diluent (0.2% v/v DMSO). Protein expression was evaluated by Western blotting (WB). Apoptosis was assayed by flow cytometry with annexin V or propidium iodide. Primary AML samples were obtained at study centers after informed consent. Isolated mononuclear cells (Ficoll) were used in colony forming unit (CFU) assays with DMSO or drug for 12-16 days. Synergy was determined by median-effect (CalcuSyn) where a combination index <1 is synergistic and >1 antagonistic. DNMT1 expression (TCGA) was highest in AML vs all other tumor types (p<0.001). In CRISPR screens (DepMap), AML cell lines were increasingly dependent on DNMT1, ATR, CHK1, and WEE1 (ANOVA p<0.001). WB of AML cell lines treated with DAC (25 - 200 nM) for 24 h showed phosphorylations of H2AX and CHK1 S296/S345 that were most pronounced in TP53MT lines. In apoptosis assays, ATRi, CHK1i, and WEE1i were synergistic with clinical concentrations of DAC (Table 1). In isogenic Molm13 lines, TP53 loss conferred DAC resistance that was overcome with combination treatment. Selective CHK1i were generally more synergistic than CHK1/2i, consistent with a CHK1 DDR. CFU assays of primary AML samples also showed synergy between DAC and ATRi, CHK1i, and especially WEE1i. Table 1. Synergy between decitabine and replication checkpoint inhibitors in preclinical models of AML. Cell line data are derived from subdiploid apoptosis assays. U937 data were replicated using annexin V. Prexasertib showed no activity in KG1a cells (suggesting drug efflux) requiring the use of the alternate CHK1 inhibitor, rabusertib. Abbreviations: ATR, ataxia telangiectasia and Rad3 related; CHK1/2, checkpoint kinase 1 and/or 2; CI, combination index; Conc, concentration; DNMT1, DNA methylatransferase 1; IQR, interquartile range; MT, mutated; ND, not determined; SD, standard deviation; WT, wild type. Drug Target Conc. (nM) Molm13 (TP53WT) Molm13 (TP53-/-) U937 (TP53G187fs*/-) KG1a (TP53V225fs*/-) Primary AML (TP53WT orTP53MT) Units Decitabine DNMT1 6.25 – 200 25 ± 11 350 ± 260 310 ± 180 6.1 ± 1.6 ND EC50, nM (mean, SD) Ceralasertib ATR 250 – 500 0.93 (0.66 – 1.24) 0.90 (0.84 – 0.96) 0.15 (0.08 – 0.21) 0.74 (0.63 – 1.02) 0.66 (0.43 – 2.43) CI with decitabine (median, IQR) Camonsertib ATR 10 – 100 0.27 (0.19 – 0.59) 0.24 (0.22 – 0.34) 0.34 (0.21 – 0.39) ND ND Prexasertib CHK1/2 1.0 – 6.0 0.89 (0.82 – 1.06) 0.78 (0.68 – 0.90) 0.95 (0.93 – 1.08) ND 0.88 (0.29 – 1.28) MK-8776 CHK1 250 – 1, 000 0.57 (0.48 – 0.80) 0.76 (0.73 – 1.19) 0.89 (0.57 – 1.39) ND ND Rabusertib CHK1 600 – 1, 000 ND ND ND 0.51 (0.48 – 0.56) ND Adavosertib WEE1 100 – 400 0.70 (0.63 – 1.11) 0.99 (0.78 – 1.14) 0.67 (0.60 – 0.74) 0.67 (0.37 – 0.93) 0.65 (0.43 – 0.88) DAC synergizes with ATRi, CHK1i, and WEE1i in AML. Future work will test these combinations in vivo and incorporate BH3 mimetics. Citation Format: Clifford M. Csizmar, Antoine N. Saliba, Olivia K. Rossman, Kevin L. Peterson, X. Wei Meng, B. Douglas Smith, Gabriel Ghiaur, Jonathan A. Webster, Mrinal M. Patnaik, Aref Al-Kali, Scott H. Kaufmann. Decitabine (DAC) induces a DNA damage response (DDR) and synergizes with replication checkpoint inhibitors in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1170.

Abstract Background: Replication checkpoint modulators (RCMs), including inhibitors of CHK1, ATR, and WEE1, have been widely studied for their ability to inhibit cell cycle checkpoints and enhance the toxicity of agents that stall replication forks. In preclinical studies each of these inhibitors also demonstrates notable activity as a single agent against relapsed/refractory acute myeloid leukemia (R/R AML). In particular, our prior study (PMID: 33414171) demonstrated that several myeloid cell lines underwent cell death at nanomolar concentrations, with a median IC50 of 9 nM irrespective of the TP53 status. Examination of pathways involved in RCM-induced leukemia cell killing demonstrated that CHK1 inhibition induces CDC25A upregulation accompanied by activation of a death program that involves a CDK2-initiated 10- to 50-fold upregulation of TNFα and downstream engagement of the extrinsic apoptotic pathway. Here we have comprehensively characterized the processes that occur between the interruption of replication checkpoints and leukemic cell death in these sensitive cells versus cells selected for RCM resistance. Methods: Using the CHK1 inhibitors MK8776 and prexasertib, ATR inhibitors berzosertib and ceralasertib, and WEE1 inhibitor adavosertib as paradigm drugs, we examined signaling in two AML cell lines, U937 and THP.1, that were selected for CHK1 inhibitor resistance, examined cross-resistance patterns, and assessed the biochemical basis for leukemic cell death. Changes in gene expression and signaling induced by RCMs were analyzed in isogenic pairs using RNA sequencing, flow cytometry and immunoblotting. Results: In contrast to parental U937 and THP.1 cells, CHK1 inhibitor-selected U937 and THP.1 cell exhibited a lack of TNFα mRNA induction and did not undergo cell death in response to multiple CHK1 inhibitors. RNA sequencing of CHK1 inhibitor-selected AML lines revealed an almost complete absence of induction of CDK2-activated genes following prexasertib treatment. Interestingly, unlike solid tumors, AML cell lines that were resistant to CHK1 inhibitors exhibited hypersensitivity to WEE1 or ATR inhibition. Further studies demonstrated that CHK1 inhibitor response was varied in primary AML samples, with 50% of primary AML specimens resistant to CHK1i monotherapy, again without any relationship to TP53 status. The common features among sensitive vs. resistant leukemias was the successful inhibition of CDK2 phosphorylation and the associated induction of TNFα. Conclusions: Our findings reveals critical new insights into the factors that influence the response to CHK1 inhibitors in AML. Importantly, we found no evidence of cross-resistance to inhibitors of other replication checkpoint proteins, enabling alternative options. Citation Format: Annapoorna Venkatachalam, Husheng Ding, Nicole Vincelette, Cordelia McGehee, Kevin Peterson, Paula Schneider, Cristina Correia, Hu Li, Scott Kaufmann. CDK2 activation mediates response of acute myeloid leukemia to CHK1, ATR and WEE1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 423.

Abstract Background: Ovarian cancer is the sixth leading cause of cancer deaths among women in the United States. South Asians in the USA, including Asian Indians and Pakistani Americans (AIPA), are a rapidly growing yet under-researched group. This study aims to analyze the clinical features, survival rates, treatment, and outcomes of ovarian cancer among AIPA women in the United States. Methods: Adult women diagnosed with ovarian cancer between 2000 and 2023 within the Surveillance, Epidemiology, and End Results (SEER) registry were identified utilizing ICD codes. The analysis focused on comparing clinical characteristics and outcomes between AIPA and Non-Hispanic White (NHW)women and excluded all women of other races/ethnicities or unknown race/ethnicity. Baseline characteristics, including age at diagnosis, disease stage, and treatment, were compared between AIPA and NHW women utilizing a chi-squared test for categorical variables and a t-test for continuous variables. The 5-year overall survival (OS) was estimated for each race/ethnicity by the Kaplan-Meier method, and the OS was further compared between AIPA and NHW in a cox-proportional hazard regression analysis adjusting for age at diagnosis, grade, histology, TNM stage, surgical debulking, completeness of debulking surgery, and receipt of chemotherapy. Similar comparisons of baseline characteristics and OS were also performed in the subset of women with high-grade serous ovarian cancer (HGSOC). Result: A total of 86, 485 women met the inclusion criteria, of which 1, 179 were AIPA and 85, 306 were NHW. AIPA had a significantly younger median age at diagnosis compared to NHW women in the overall population (57 vs. 65 years, p<0.001) and among 448 AIPA and 29, 677 NHW women with HGSOC (58 vs. 65 years, p<0.001). Approximately two-thirds of women in both groups presented with Stage III or IV ovarian cancer at diagnosis and had a serous subtype of ovarian cancer. Similarly, after the exclusion of unknown grade, high-grade tumors accounted for more than two-thirds (75 vs. 72%) of the ovarian cancer cases in both groups. AIPA were more likely to undergo debulking surgery (85 vs. 80%, p<0.001) and receive chemotherapy (77 vs. 72%, p<0.001) compared to NHW women. The unadjusted 5-year OS for AIPA and NHW women were 56% vs. 43% in the overall population, and 53% vs. 41% among women with HGSOC, respectively. In multivariate analysis of the entire study population, AIPA women had a significantly better OS than NHW women (HR: 0.86, 95%CI: 0.79 - 0.94, p<0.001). Among women with HGSOC, a similar trend of better OS was noted in multivariate analysis among AIPA women compared to NHW (HR: 0.88, 95%CI: 0.77 - 1.01, p=0.07), although this did not reach statistical significance. Conclusion: The younger age at diagnosis, along with a generally better prognosis, among AIPA women compared to NHW women, suggests a possible enrichment of DNA-repair gene alterations among AIPA women. Citation Format: Aditi Karki, Sumeet Kumar Yadav, Muhammad Rafiqul Islam, Raja Pramanik, Ramila Shilpakar Dangol, Zeba Aziz, Sandhya Pruthi, Maryam Shahi, Jamie N. Bakkum-Gamez, Scott H. Kaufmann, Fergus J. Couch, Siddhartha Yadav. Clinical characteristics and survival outcomes of ovarian cancer among South Asian women in United States [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7118.

Breast and ovarian cancer represent the most common and lethal cancers in women. Despite chemotherapy and targeted agents, recurrence is frequent and outcomes for patients with advanced disease are dismal, underscoring the need for novel therapeutic strategies. Using an unbiased approach with DepMap CRISPR screens across more than 1000 cancer cell lines, we identified six genes (CTPS1, RHOA, PRKRA, RAD9A, HUS1, and RAD1) that were essential for triple negative breast cancer (TNBC) cells, but not ERα+ breast cancer cells, that were also associated with poor outcomes. Validation of these effects by siRNA knockdown studies revealed that suppression of CTPS1 elicited the most potent inhibitory effects, characterized by S phase cell cycle arrest and apoptosis. Similar effects were also found in ovarian cancer cells which share many molecular features. CTPS1, along with CTPS2, is responsible for enzymatic conversion of UTP to CTP, an essential nucleoside triphosphate required for multiple cellular processes including DNA/RNA synthesis, phospholipid synthesis and glycosylation. CTPS1 was found to be highly expressed in tumor tissues, for both breast and ovarian cancer. RT-PCR assessment of CTPS1 expression in a panel of over 40 breast and ovarian cancer lines confirmed variable, but elevated expression relative to normal models. Notably, chemotherapy-resistant and PARP inhibitor (PARPi)-resistant models exhibited the highest levels of CTPS1 expression among all cells analyzed. In partnership with Step Pharma, which has developed a first-in-class highly selective CTPS1 inhibitor (STP938), we have demonstrated that STP938 elicits potent anti-proliferative effects at nM concentrations across many cell line and patient derived models, both in 2D and 3D culture systems. Furthermore, STP938 exhibited strong anti-cancer activity in chemotherapy- and PARPi-resistant ovarian cancer models, both as a monotherapy and in combination with standard-of-care treatments. RNA-seq analysis in cell models following STP938 treatment or CTPS1 knockdown/knockout showed significant dysregulation of DNA replication and cell cycle related genes, suggesting potential synergy with drugs targeting replication stress or DNA damage response pathways. Single-cell sequencing further revealed specific cell-cell interactions/cross talk/pathways that are disrupted by STP938 in vivo using ovarian cancer patient-derived xenograft. The first-in-human clinical trial for STP938 began in 2022 for relapsed/refractory B- and T-cell lymphomas (NCT05463263). In August 2024, a second phase I trial opened for advanced solid tumors, including an expansion cohort for CTPS2-null ovarian cancer (NCT06297525) at Mayo Clinic. These laboratory discoveries, and ongoing clinical studies, are expected to pave the way for a broader uptake of this novel therapeutic approach aimed at improving outcomes in patients with highly aggressive forms of these diseases. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Epp, Esther Rodman, Noa Odell, Nicole Pearson, Xiaonan Hou, Matthew Goetz, Scott Kaufmann, John Weroha, Philip Beer, John Hawse. Exploiting CTPS1 dependency for the treatment of breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6388.

ABSTRACT In our efforts to enhance sensitivity to PARP inhibitors, we identified clofarabine (CLF) as a potential therapy for drug-resistant ovarian cancer and nuclear trafficking of Cathepsin L (CTSL) as a treatment- responsive biomarker. Using PARP inhibitor-sensitive and -resistant OC cell lines, ex vivo cultures of patient-derived ovarian ascites (OVA), primary ovarian tumors, and xenografts (PDX), we found that CLF monotherapy induces nuclear CTSL (nCTSL) in CLF-responsive cells (CLF-r) and sensitizes them to PARP inhibitors olaparib and rucaparib. In CLF non-responsive cells (CLF-nr), a combination of CLF with olaparib is necessary for nCTSL trafficking and synergy. CLF+olaparib synergy was observed in 47% of CLF-r and 24% of CLF-nr OVA samples. Drug-induced nCTSL is crucial for DNA damage response, including cell cycle arrest and apoptosis. Knockdown of CTSL in both CLF-r and CLF-nr cells conferred resistance to the CLF+olaparib combination, emphasizing nCTSL’s role in the DNA damage response pathway (DDR). Mechanistically, CLF facilitates CTSL nuclear import via KPNB1 in CLF-r cells. In CLF-nr cells, both olaparib and CLF are needed to facilitate CTSL nuclear import. Additionally, CLF downregulates the nuclear export protein CRM1 (XPO1) in both cohorts. Interestingly, CLF does not downregulate CRM1 in a subset of OVAs (29%), and they were classified as CLF-resistant (CLF- Res). In these samples, inhibiting CRM1 with KPT8602 restored synergy between CLF and PARP inhibitors. In vivo, CLF-r and CLF-nr PDX models exhibited enhanced DDR, reduced tumor burden, and prolonged survival with the CLF+olaparib combination. These findings suggest the CLF+olaparib combination is a promising therapeutic strategy for drug-resistant OC by inducing DDR through CTSL nuclear localization.

Introduction Acute myeloid leukemia (AML) with mutated TP53 (TP53MT) is resistant to chemotherapy and has a poor prognosis. Prior reports suggest that DNA methyltransferase inhibitors (DNMTi), such as decitabine and azacitidine, form covalent DNA-DNMT1 adducts and invoke a DNA damage response (DDR) characterized by activation of the ATR-CHK1 pathway, including in TP53MT AML samples. This study evaluates whether decitabine exhibits cytotoxic synergy with replication checkpoint modulators (ATRi, CHK1/2i, WEE1i) in pre-clinical models of AML. Methods Parental cell lines (U937, KG1a, Molm13, MV-4-11) were obtained commercially and genomically validated. The Molm13 TP53-/- cell line was generated via CRISPR/Cas9 knockout. Cells were maintained in RPMI-1640 medium with 10% FBS, 2 mM glutamine, and antibiotics at 37 ºC, 5% CO2. Decitabine, ceralasertib (ATRi), prexasertib (CHK1/2i), MK-8776 (CHK1i), and adavosertib (WEE1i) were reconstituted at 10 mM in DMSO. For drug treatments, cells were cultured for 48 h in medium with 0.2% (v/v) DMSO or drug. Western blots were stained with commercial antibodies followed by peroxidase-coupled secondary antibodies and imaged by chemiluminescence. Apoptosis assays were performed via flow cytometry (BD FACSCanto II) with annexin V-APC (BD Biosciences) and/or propidium iodide (Sigma) staining. Peripheral blood and bone marrow samples were obtained from AML patients after informed consent at Mayo Clinic and Johns Hopkins University. Mononuclear cells were isolated via Histopaque-1077 (Sigma) centrifugation and used immediately. Colony forming assays were performed in Methocult H4435 (Stem Cell Technologies) supplemented with vehicle or drug; leukemic colonies were counted after 12-16 days. Combination indices (CI) were determined by the Chou-Talalay method (CalcuSyn) where values <1 indicate synergy and >1 antagonism. This work was supported in part by R01 CA289285. Results Western blots of TP53MT AML cell lines (U937, KG1a) and primary samples treated with decitabine (≤200 nM) for 24 h demonstrated increased phosphorylation of H2AX and S296/S345 CHK1, consistent with activation of the ATR-CHK1 DDR pathway. These changes were less pronounced in TP53 wild type cell lines (Molm13, MV-4-11). In subdiploid (SubG1) assays, U937 cells exhibited resistance to monotherapy with decitabine (EC50 = 330 ± 200 nM) and variable sensitivity to ceralasertib (250 - 500 nM), prexasertib (1 - 10 nM), MK-8776 (250 - 1,000 nM), and adavosertib (50 - 400 nM). When combined, however, marked synergy was observed between decitabine and both ceralasertib (mean CI 0.15) and adavosertib (CI 0.59). Synergy was also observed with the selective CHK1i, MK-8776 (CI 0.56), but less-so with the dual CHK1/2i, prexasertib (CI 0.93), consistent with the hypothesis of an ATR-CHK1 DNA damage response. Similar results were obtained from decitabine (EC50 = 300 ± 100 nM) and ceralasertib (CI 0.54), adavosertib (CI 0.67), MK-8776 (CI 0.52), and prexasertib (CI 0.81) in annexin V assays. These combinations were further evaluated (SubG1 assays) in an isogenic pair of Molm13 and Molm13 TP53-/- cell lines. In Molm13 cells, decitabine (EC50 = 23 ± 11 nM) was again synergistic with adavosertib (CI 0.60), but not ceralasertib (CI 1.3) or prexasertib (CI 1.2). In Molm13 TP53-/- cells, however, very little synergy was observed between decitabine (EC50 = 53 ± 22 nM) and ceralasertib (CI 1.5), prexasertib (CI 4.3), or adavosertib (CI 1.0) except for at higher concentrations of adavosertib (400 nM, CI 0.89). Thus, while biallelic TP53 inactivation conferred resistance to DNMTi and checkpoint inhibitors, the degree of resistance was context-dependent and could be partially overcome by WEE1 inhibition. The combinations of decitabine with ceralasertib (n = 2), prexasertib (n = 4), or adavosertib (n = 3) were evaluated in colony forming assays using primary AML samples from newly-diagnosed patients with diverse clinicomolecular profiles. Combination treatment was antagonistic with ceralasertib (CI 2.2), additive with prexasertib (CI 1.0), and synergistic with adavosertib (CI 0.88). Conclusions In multiple cell lines and diverse primary AML samples, decitabine activated the ATR-CHK1 DDR pathway and synergized with ATRi, CHK1/2i, and WEE1i. Amongst these, only the combination of decitabine plus adavosertib exhibited synergy across all three models, highlighting WEE1 as a potential target in TP53MT AML.

TPS5630 Background: Platinum-resistant ovarian cancer (PROC) portends a poor prognosis and, although treatments are available, response rates are low. Previous studies have demonstrated that DNA topoisomerase I (TOP1) inhibitors exhibit clinical activity in PROC. Irinotecan also has a 15-25% response rate in some studies of platinum-resistant epithelial ovarian cancer but does not have FDA approval for this indication. Additional studies in preclinical models suggest that TOP1 poisons are more active if administered at low doses on prolonged schedules. PLX038 is a long-acting prodrug of the potent TOP1 inhibitor SN-38. In PLX038, SN-38 is covalently conjugated to circulating polyethylene glycol and is slowly released at a constant rate to provide the active agent with a long half-life, a low Cmax and very high exposure – important facets for optimal safety and efficacy of this drug. Further, in preclinical studies the nanoparticle prodrug accumulates and is retained in tumors, where it slowly releases the SN-38. Methods: This single arm, phase 2 clinical trial is open at Mayo Clinic in Rochester, MN. The primary endpoint is overall response rate and secondary endpoints include progression-free survival and assessment of tolerability of PLX038. Correlative goals include i) measurement of TOP1-DNA covalent complexes in tumor biopsies and circulating tumor cells to assess successful target engagement; ii) assessment of homologous recombination status and SLFN11 expression for correlation with tumor response rate; and iii) pharmacokinetics of SN-38 and SN-38G as well as their association with GI toxicity. Assessment of gut microbiota and association with GI toxicity profile are also being investigated. Eligible patients include women with platinum resistant high grade serous ovarian, primary peritoneal, and fallopian tube cancer who have received fewer than two prior chemotherapy regimens in the platinum-resistant setting. All patients must have an ECOG performance status of 0, 1, or 2 and adequate bone marrow, renal, and hepatic function. As of January 31, 2024, 12 of planned 37 patients have enrolled. Clinical trial information: NCT05465941 .

Abstract Purpose: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). Experimental Design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial–mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

Abstract Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPN) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for patients with triple-negative (TN) myelofibrosis (MF) who lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in TN-MF and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves numerous hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC–alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. Significance: This study establishes that MYC expression is increased in TN-MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in TN-MPNs.