Patnaik, Mrinal M.

Background Clonal hematopoiesis (CH) is defined by somatic mutations in hematopoietic stem cells, often conferring a selection advantage based on clonal selection pressures. CH in DNA Damage Response and Repair (DDR, e.g. TP53, PPM1D) genes are frequent in individuals exposed to chemotherapy and radiation (chemo/XRT). In metastatic prostate cancer (mPC) patients, the prevalence of CH is estimated at 57% and is increased among those with prior exposure to chemo or XRT. In these patients, the use of radiotheranostics is becoming more prevalent and are now being tested in earlier lines of therapy. We have identified an increase in DDR-somatic variants post lutetium-177-PSMA, raising concern for long term hematologic safety. Here, we evaluate the spectrum of CH and CCUS (clonal cytopenias of undetermined significance) in mPC patients, prior to receiving radioisotope therapies. Methods We performed error corrected next-generation sequencing (NGS) on peripheral blood samples from 67 mPC patients enrolled in the Prostate Cancer Medically Optimized Genome Enhanced Therapy Study (PROMOTE) study (NCT #01953640). All patients enrolled also had whole exome sequencing (WES) performed on initial metastatic biopsies and on whole blood for germline genotyping. Somatic variants with a variant allele fraction (VAF) ≥0.5% were considered as CH, using standard bioinformatic variant calling algorithms. Results The median age at NGS was 74.4 years (range, 58.4-92.3). While all patients received androgen deprivation therapy (ADT) +/- androgen receptor pathway inhibitor (ARPI), 67.2% (n=45, pelvic XRT- 35, palliative XRT- 10) received prior XRT and 3% (n=2) received prior docetaxel. No patients in this cohort received prior platinum or PARP inhibitor. Of those treated with XRT or chemo, the median time from first exposure to CH sample collection was 4.1yrs (range, 0.12-29.6) and 2.3yrs (range, 0.6-4.0), respectively. Of the 67 patients, we identified a total of 111 pathogenic variants in 59 (88.1%) patients. We compared our NGS data to the germline data and tumor for each patient (All samples obtained at the same time point; Wang et al 2018). Our analysis excluded 2 ATM variants likely to be germline, and 1 TP53 variant likely to be tumor-derived. Each patient had at least one somatic variant, with a median of 2 variants (range, 1-6The median CH VAF was 1.8% (range, 0.5-50.2) with 91.0% of alterations detected at a VAF of <10%. Specific somatic variants, presence/absence of CH, number of variants, nor VAF was associated with age, prior chemo, or XRT exposures. At time of NGS, 54% of patients were anemic (hgb <13g/dL), 7% were thrombocytopenic (platelets <135 x109), 6% were leukopenic (WBC <3 x 109), and no patients were neutropenic (ANC <1 x109). Persistent cytopenias, lasting >3months, were present in 25% (n=17) of patients; however, bone marrow biopsies (BMB) were performed only in 6% (n=4). Three (75%) of these 4 patients had mPC infiltration in the BM, and 1 (25%) had mild dysplasia not meeting criteria for MDS but meeting criteria for CCUS (CHEK2 somatic variant). At last follow-up, there were no patients formally diagnosed to have myeloid neoplasm (MN). Median overall survival (OS) from prostate cancer diagnosis was 8.6yrs (range, 1.4-28.4). With a median follow up of 2.2yrs (range 0.1-10.8) after CH testing, 3.0% (n=2) were alive, with most deaths occurring due to mPC. The presence of CH did not impact overall survival (OS), however, presence of cytopenias at the time of NGS testing was associated with worse outcomes (median OS 2.5yrs vs 1.2 yrs., p=0.025). Conclusions We define the landscape of CH mutations in mPC patients predominantly treated with ADT/ARPI and XRT, demonstrating that DNMT3A, TET2, PPM1D and ASXL1 are frequently mutated (88%) and largely subclonal (91% with VAF ≤10%). While 25% of these patients had cytopenias, with cytopenias negatively impacting survival, due to inadequate BM biopsy work up, the true prevalence of CCUS and MN could not be estimated. Given the advancement of genotoxic therapies and the high prevalence of subclonal CH in mPC, future studies investigating CH as a predictive biomarker of hematological dysfunction are much needed.

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.

Background: Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative overlap neoplasm (MDS/MPN), further categorized as MD (WBC < 13 x 109/L) and MP (≥ 13 x 109/L) subtypes with an inherent risk for AML transformation (Am J Hematol 2024; 99: 1142). Hypomethylating agents (HMA), decitabine (DAC) and azacitidine, are the only FDA approved agents for the management of CMML, with complete remission rates of <20%, and a recent prospective study showing no event free survival benefit of DAC over hydroxyurea in MP-CMML (J Clin Oncol 2023; 41: 1888). TET2 mutations (TET2MT) occur in approximately 60% of CMML cases. TET2 converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), resulting in DNA demethylation (Nat Rev Gen 2017; 18: 517). IV ascorbic acid (AA) at pharmacologic concentrations demonstrates anti-cancer activity via H2O2-induced oxidative stress and DNA demethylation mediated by TET-activation. AA, a cofactor of TET2, binds to its catalytic domain, restores, and enhances TET1/2/3 activity. Given the synergistic impact of IV AA and HMA on DNA methylation, we designed a phase II clinical trial testing the safety and efficacy of standard dose decitabine and high dose IV AA in HMA-naïve CMML patients. Methods: LS1781 (NCT03418038) is an investigator-initiated, prospective, single-arm, single-institution, phase II trial assessing the safety and preliminary efficacy of high-dose IV AA in combination with DAC for patients with TET2MT CMML. DAC 20 mg/m2 Days 1-5 of each 28-day cycle is given as standard of care. With each cycle, IV AA (1g/kg, maximum 100g) is given 3 times on Days 1, 3, 5 via central line within 2 hours after DAC. Patients are encouraged to take oral AA 1000 mg once daily on non-treatment days. The primary endpoint is overall response rate determined by IWG MDS/MPN 2015 criteria after 4 cycles of therapy. Secondary endpoints include safety and side effects. Exploratory endpoints include the impact of AA on TET2MT variant allele fraction, plasma cytokine levels, DNA methylation and hydroxymethylation (as measured by Infinium MethylationEPIC array), and hematopoietic progenitor colony forming assay assessment (colony counts and impact on differentiation). Inclusion criteria include patients ≥ 18 years with TET2MT CMML, ≤1 cycle of HMA or ESA (hydroxyurea allowed), platelets ≥ 20,000/m3, ANC ≥ 500/mm3, and Cockcroft-Gault creatinine clearance ≥ 60 mL/min/m2. Twenty-three patients will be enrolled at Mayo Clinic Rochester and the Mayo Clinic Health System, based on power calculations to demonstrate enhanced complete response and overall response rates, in comparison to those seen historically with decitabine alone. Dose modifications have been established and will be considered based on adverse events. Treatment will be discontinued for disease progression or in the event of ≥ Grade 3 adverse events that do not resolve promptly. After completion of up to 12 cycles, patients with stable disease or better may continue single agent DAC with or without oral AA. Enrollment to the trial began in February 2024, with 2 patients enrolled at the time of abstract submission.

Abstract DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematologic neoplasms. Although individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here, we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome [ARDS]) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant (DNMT3Amt) CH on COVID-19–related ARDS and mortality. Using single-cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single-cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of interleukin-32 (IL-32), a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single-cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19–related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19.

Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy with limited therapeutic options. Single-cell analysis of clonal architecture demonstrates early clonal dominance with few residual WT hematopoietic stem cells. Circulating myeloid cells of the leukemic clone and the cytokines they produce generate a deleterious inflammatory climate. Our hypothesis is that therapeutic control of the inflammatory component in CMML could contribute to stepping down disease progression. The present study explored the contribution of immature granulocytes (iGRANs) to CMML progression. iGRANs were detected and quantified in the peripheral blood of patients by spectral and conventional flow cytometry. Their accumulation was a potent and independent poor prognostic factor. These cells belong to the leukemic clone and behaved as myeloid-derived suppressor cells. Bulk and single-cell RNA-Seq revealed a proinflammatory status of iGRAN that secreted multiple cytokines of which CXCL8 was at the highest level. This cytokine inhibited the proliferation of WT but not CMML hematopoietic stem and progenitor cells (HSPCs) in which CXCL8 receptors were downregulated. CXCL8 receptor inhibitors and CXCL8 blockade restored WT HSPC proliferation, suggesting that relieving CXCL8 selective pressure on WT HSPCs is a potential strategy to slow CMML progression and restore some healthy hematopoiesis.

Abstract Drug development for chronic myelomonocytic leukemia (CMML) has failed to parallel the recent success observed in related myeloid neoplasms. To address these shortcomings, the US Food and Drug Administration (FDA) held a “Mini-symposium on CMML: Current State of the Art and Trial Design” in September 2023. This symposium brought together a panel of key FDA regulators and academic experts in CMML drug development to discuss challenges and provide perspectives on future drug development for this disease. The panel explored unique challenges that underlie the lack of therapeutic advances in CMML to date and discussed relevant topics such as clinical trial design, study end points, and key regulatory considerations. This article summarizes the key points of discussion from this symposium to facilitate advancements in the field.

Abstract Objectives To define the prevalence, distribution and characteristics of patients with VEXAS (vacuoles, E1-enzyme, X-linked, autoinflammation, somatic) syndrome who have confirmed vasculitis. Methods Patients with VEXAS syndrome, verified by positive UBA1 mutation, were included. Chart review was performed to identify patient characteristics and outcomes. Vasculitis diagnosis was based on either histopathology showing vascular inflammation or non-invasive angiography findings. Summary statistics were calculated. Results Eighty-nine patients met inclusion criteria. All were male with a median age of onset of 66.9 years (interquartile range 60.1, 72.7). Median (interquartile range) follow-up was 3.8 (2.2–5.5) years, during which 21 patients (23.6%) had evidence of vasculitis. Vasculitis subtypes included small vessel vasculitis (19.1%), cutaneous medium vessel vasculitis (2.2%) and large vessel vasculitis (2.2%). No patient had more than one vessel size involved. Histopathology in small vessel vasculitis patients was consistent with cutaneous leukocytoclastic vasculitis in the majority, though one patient had leukocytoclastic peritubular capillaritis on renal biopsy. Cranial symptoms (headache, vision changes or jaw pain) were noted in 18.0%. Two additional patients not experiencing cranial symptoms exhibited large vessel involvement with confirmed carotid thickening on non-invasive angiography; one of these had a positive temporal artery biopsy. Conclusion VEXAS syndrome manifests as a variable vessel vasculitis in a quarter of patients, with cutaneous small and medium vessel involvement being particularly common. Some patients may have positive ANCA serologies or even renal vasculitis leading to misdiagnosis. Cranial symptoms are common and may mimic GCA, though documented large vessel inflammation is rare.

Abstract This phase 2 trial assessed high-dose IV ascorbic acid in TET2 mutant clonal cytopenia. Eight of 10 patients were eligible for response assessment, with no responses at week 20 by International Working Group Myelodysplasia Syndromes/Neoplasms criteria. This trial was registered at www.clinicaltrials.gov as #NCT03418038.