Bulk RNA-Seq data is widely used to identify differentially expressed genes (DEGs) between groups of samples. Typically, genes with missing values are discarded in initial analyses, resulting in a loss of information. Although only a few studies apply imputation, no single method is considered the gold standard, as appropriate choice of imputation should ideally depend on the underlying cause of missingness. But identifying this cause is often challenging. Consequently, many studies rely on complete-case (CC) analysis due to its simplicity, but that introduces bias. Our simulations show that while CC controls false-positives, it lacks sensitivity, leading to under detection of truly disease-associated genes. Moreover, simple mean or median imputations may also result in bias when missing data arises from biological or technical factors, which is often a possibility in RNA-Seq data. As expression of a gene may correlate with co-regulated genes, missing gene counts could be imputed based on similarly expressed genes. The concept of imputing missing values in K-nearest neighbor (KNN) imputation algorithm aligns aptly with this biological phenomenon. If a gene count is missing, KNN imputation estimates this value using expression data from K other genes with similar expression patterns. In this article, using simulations we show that common imputation techniques for detecting DEGs perform better than CC analysis under varying percentages of missing data. On application to in-house real-data on Acute Myeloid Leukemia, we identified that compared to CC, common imputations detect more DEGs. Additionally, we provide recommendations for handling genes with low counts across all samples.

Acute myeloid leukemia (AML) is the most common and lethal leukemia in adults. AML consists of many genetic subtypes, which limits broad applicability of targeted therapy. We discovered that the hematopoiesis-restricted tetraspanin CD37 is expressed on the majority of primary AML blasts and thus may represent a common therapeutic target for AML regardless of subtype. We demonstrate that the internalization properties of CD37 are distinct in AML blasts when compared with normal blood cells, and that CD37 rapidly accumulates inside AML blasts via dynamin-dependent endocytosis. Our work revealed that the clinically relevant anti-CD37 antibody-drug conjugate (ADC) Debio 1562 (alpha CD37DM1) is highly cytotoxic to AML blasts, but not normal hematopoietic stem cells. We found that alpha CD37-DM1 improved clinical outcomes and overall survival in multiple in vivo models of AML. Together, these data demonstrate that targeting CD37 with an ADC such as alpha CD37DM1 is a feasible and promising therapeutic option for the treatment of AML.

Acute graft-versus-host disease (GVHD) is a donor T cell driven complication and the leading cause of non-relapse mortality in patients receiving an allogeneic hematopoietic cell transplantation (allo-HCT). Allogeneic donor T cells eradicate residual leukemia and prevent relapse via the graft-versus-leukemia (GVL) effect and are critical for responding against opportunistic infections post-transplant. Current regimens successful in preventing GVHD are broadly immunosuppressive and come at the cost of increased risk of relapse and/or infection. Therefore, there is an urgent need for new approaches that limit GVHD while retaining GVL responses. During GVHD, alloreactive T cells boost their energy production through oxidative phosphorylation (OXPHOS) and glycolysis, supporting heightened proliferation and pathogenicity against healthy host tissues. The enzyme dihydroorate dehydrogenase (DHODH), is essential for de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential during OXPHOS. Having shown upregulation of DHODH messenger RNA and protein expression in activated human T cells, we evaluated DHODH inhibition, via a small molecule inhibitor HOSU-53, as a therapeutic approach for GVHD. Inhibiting DHODH significantly reduced oxidative metabolism in T cells both during and after activation, while selectively suppressing inflammatory cytokine production in de novo activated, but not previously activated, T cells. In a xenogeneic model, HOSU-53 treatment limited GVHD severity, decreased pathogenic Th1 and Th17 response, and preserved beneficial GVL effects. Altogether, we identify DHODH inhibition as an innovative treatment strategy in allo-HCT recipients to reduce GVHD severity and retain effective GVL response.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by expansion of mutant hematopoietic stem and progenitor cells (HSPCs) and an increased risk of chronic diseases and cancers. While mutations in DNMT3A , TET2 , and ASXL1 are common in CHIP, the contribution of less frequent gene mutations is not well understood. Here, we report MYD88 mutations, including lymphoma-associated and novel variants in blood cells of the general population and newly diagnosed solid cancer patients. MYD88 CHIP mutations in HSPCs activate NF-κB, indicating a gain-of-function activity. Modeling MYD88 CHIP in mice, Myd88 L252P (equivalent of human L265P) expression resulted in a competitive fitness advantage of HSPCs. Myd88 L252P HSPCs exhibit a myeloid cell bias and inflammation, leading to hematologic disease. Single-cell RNA sequencing indicated that Myd88 L252P expands distinct hematopoietic and immune cell clusters and activates immune-related pathways in HSPCs. An IRAK1/4 inhibitor suppressed MYD88-dependent NF-κB activation and reversed Myd88 L252P cell expansion. Overall, MYD88 mutations contribute to CHIP by inducing innate immune pathways in HSPCs and inflammatory disease.

Small-cell lung cancer is an aggressive subtype of lung cancer with poor prognosis and poor overall survival and comprises approximately 15% of all lung cancers. SCLC, unlike NSCLC, has no known targetable driver mutations and, therefore, targeted therapeutics have been lacking. The frontline therapy for SCLC is platinum-etoposide chemotherapy, a regimen that has been in use for 30 years. Although patients respond well to this combination initially, they almost always relapse shortly after the start of therapy and second line therapies typically provide only a few months of benefit. Clearly, better therapeutics are necessary for the treatment of SCLC. Cancer cells often have dysregulated metabolic pathways. These changes are often necessary to enable the continued proliferation of the cancer cells. Therefore, key metabolic enzymes can have a crucial role in tumor cells survival and can be considered targets for cancer treatment. According to some recent studies, dihydroorotate dehydrogenase (DHODH), which catalyzes the conversion of dihydroorotate to orotate in the pyrimidine de novo synthesis pathway, has a particular role in the survival of different types of cancers including leukemia and SCLC. We evaluated the effect of HOSU-53, a novel DHODH inhibitor, on SCLC tumor growth in vitro and in vivo. We measured IC50s of 18 SCLC cell lines in vitro using CellTiter-Glo® Luminescent Cell Viability Assay and most SCLC cells showed sensitivity to HOSU-53 treatment in the low nanomolar range. Additionally, to determine the role of salvage pathway in HOSU-53 sensitivity, we treated the sensitive and resistant cells in parallel with HOSU-53 alone or in combination with exogenous uridine. In the presence of uridine, cell viability was significantly rescued in resistant cells compared to HOSU-53 treatment as single agent. However, we did not observe rescued cell viability in sensitive SCLC cells. These results suggesting that in SCLC cells which are resistant to de novo pyrimidine inhibition, activation of salvage pathway may be one of the compensatory mechanisms for tumor cells to sustain their survival. To evaluate the effect of DHODH inhibition on SCLC tumor growth in vivo, we treated xenograft mice models with HOSU-53 alone or in combination with etoposide plus cisplatin as the standard chemotherapy for SCLC. We observed a significant decrease of tumor volume when HOSU-53 was used alone and in combination, without significant animal weight loss. Together, these data demonstrated the promising efficacy of HOSU-53 in different SCLC subtypes and support the strategy of targeting DHODH as a potential therapy to treat SCLC. These findings support the initiation of a phase l clinical trial to evaluate the preliminary efficacy and tolerability of HOSU-53; therefore, we have initiated a first-in-human phase I trial of this agent. Bahareh Nourmohammadi, Ola A. Elgamal, Sandip Vibhute, Christopher C. Coss, Thomas E. Goodwin, Erin Hertlein, Joseph M. Amann, Chad E. Bennett, John C. Byrd, David P. Carbone. Promising therapeutic effects of pyrimidine synthesis inhibition by a novel dihydroorotate dehydrogenase inhibitor in small cell lung 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 6902.

Despite recent efforts to elucidate the dependence of acute myeloid leukemia (AML) on the bone marrow microenvironment, the contribution of aged bone marrow mesenchymal stem cells (BMMSCs) to AML patient survival and treatment response remains understudied. Deregulated nutrient sensing is an established hallmark of aging and previous works have identified age-dependent metabolic dysregulations in cancer. The dependence of AML on oxidative phosphorylation (OXPHOS) has been well established, as AML adapts to fluctuations of nutrient and oxygen availability in the bone marrow microenvironment. We hypothesized that metabolic pathways, such as oxidative phosphorylation, are critical in aged AML-BMMSCs as they drive pro-tumorigenic properties and drug resistance. Our group expanded primary Normal Donor (ND)-BMMSCs and AML-BMMSCs (n=9 CBF-AML, n=11 CK-AML, n=7 ND-BMMSCs) under conditions that mimic the bone marrow microenvironment (91% nitrogen, 4% oxygen, and 5% CO2). We isolated DNA and RNA from early passaged cells to perform methylation analyses using 850k Illumina DNA methylation arrays and gene expression analyses using mRNA-seq. We utilized the Horvath Skin and Blood clock to calculate epigenetic age based on DNA methylation. qPCR assessing mRNA expression of OXPHOS-related genes in ND- and AML-BMMSCs was performed to validate sequencing results. Western blot analyses of Total OXPHOS and its complexes were performed to assess expression levels of OXPHOS-related proteins in ND- and AML-BMMSCs. Seahorse assays were performed to obtain live and repeated metabolic measurements of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in ND- and AML-BMMSCs. Lastly, 2D co-culture experiments were performed to assess cell viability of AML cell lines with BMMSCs treated ± OXPHOS inhibitors (IACS-010759 and Tamoxifen) utilizing AO/PI staining. Our integrative omics approach showed that with increased epigenetic age, as measured by the Horvath Skin and Blood clock, AML-BMMSCs reveal upregulations in hallmark gene sets for MYC and OXPHOS when compared to ND-BMMSCs. Western blot analyses confirmed upregulation of MYC and OXPHOS complexes in AML-BMMSCs when compared to controls. Results from seahorse assay revealed an increase in OCR and ATP production in AML-BMMSCs when compared to controls. Lastly, our 2D co-culture experiments in vitro revealed a decrease in cell viability on AML cells that were co-cultured with AML-BMMSCs treated with IACS-010759 and/or Tamoxifen. Our results elucidate age-dependent metabolic dysregulations within the bone marrow microenvironment in AML and provide novel targetable approaches. Future portions of testing aim to identify the mechanism of action of AML-BMMSCs and OXPHOS. Mary E. Fuentes, Julia K. Christian, Amy Kovacs, James R. Lerma, Liang Niu, Alice S. Mims, Christopher C. Oaks, John C. Byrd, Erin K. Hertlein, Amina Abdul-Aziz. Metabolic dysregulations of aged bone marrow mesenchymal cells in Acute Myeloid Leukemia reveal novel therapeutic targets [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 1542.

Genomic profiles and prognostic biomarkers in patients with acute myeloid leukemia (AML) from ancestry-diverse populations are underexplored. We analyzed the exomes and transcriptomes of 100 patients with AML with genomically confirmed African ancestry (Black; Alliance) and compared their somatic mutation frequencies with those of 323 self-reported white patients with AML, 55% of whom had genomically confirmed European ancestry (white; BeatAML). Here we find that 73% of 162 gene mutations recurrent in Black patients, including a hitherto unreported PHIP alteration detected in 7% of patients, were found in one white patient or not detected. Black patients with myelodysplasia-related AML were younger than white patients suggesting intrinsic and/or extrinsic dysplasia-causing stressors. On multivariable analyses of Black patients, NPM1 and NRAS mutations were associated with inferior disease-free and IDH1 and IDH2 mutations with reduced overall survival. Inflammatory profiles, cell type distributions and transcriptional profiles differed between Black and white patients with NPM1 mutations. Incorporation of ancestry-specific risk markers into the 2022 European LeukemiaNet genetic risk stratification changed risk group assignment for one-third of Black patients and improved their outcome prediction. Analysis of exomes and transcriptomes from 100 African American patients with acute myeloid leukemia identifies ancestry-related variation in mutation profiles and survival. Refined risk classification suggests clinical relevance of these ancestry-associated differences.

Clonal hematopoiesis (CH) is a prevalent age-related condition wherein somatic mutations accumulate in hematopoietic stem and progenitor cells (HSPCs), resulting in the clonal expansion of mutated cells. CH has been causally associated with an increased risk of cardiovascular diseases and blood cancers. While mutations in certain genes, such as DNMT3A and TET2, account for the majority of CH cases, many somatic mutations in other genes remain less studied, and their contributions to CH are unknown. We sought to characterize novel drivers of clonal expansion by examining recurrent mutations in publicly available sequencing data and newly identified CH individuals. We describe recurrent MYD88 mutations, including the lymphoma-associated mutation L265P, alongside newly discovered mutations at T71I, R140Q, and C203R, identified in the mononuclear cells of newly diagnosed solid tumor cancer patients. MYD88 is a critical adaptor in innate and inflammatory signaling. Upon activation of the Toll-like receptor (TLR) superfamily, MYD88 recruits IRAK family members (IRAK1 and IRAK4), leading to the activation of NF-kB, MAPKs, and other inflammatory pathways. Unlike wild-type MYD88, we found that the expression of the MYD88 CH mutations in myeloid hematopoietic cells results in the activation of NF-kB signaling without TLR stimulation, suggesting that the L265P, T71I, R140Q, and C203R mutations lead to MYD88 gain-of-function activity, similar to the previously reported MYD88 L265P mutation in human lymphoma. Moreover, the expression of Myd88 L252P (equivalent of L265P in humans) in mouse cKit+ bone marrow (BM) cells resulted in elevated NF-kB activation. These findings indicate that MYD88 CH mutations exhibit gain-of-function activity and result in NF-kB signaling in HSPCs. To determine whether the expression of MYD88 CH mutations in HSPCs results in a clonal fitness advantage, we first examined colony-forming potential in methylcellulose using the conditional Myd88 L252P mouse model (under the control of RosaCreERT2 inducible reporter). Expression of Myd88 L252P in mouse cKit+ BM cells resulted in increased colony replating potential compared to WT cKit+ BM cells. Moreover, in competitive BM transplantation assays, the expression of Myd88 L252P conferred a competitive advantage in the PB and BM compared to WT cells. Examination of BM and PB by flow cytometry revealed that Myd88 L262P HSPCs exhibited myeloid-biased hematopoiesis, including increased monocytes and neutrophils. Furthermore, the expression of Myd88 L262P in hematopoietic cells led to a hematologic disease without evidence of lymphoid malignancies. These observations revealed that MYD88 CH mutations lead to a competitive fitness advantage of BM cells, consistent with the more common CH mutations, such as in TET2 and DNMT3A. Given that active MYD88 mutations are associated with dysregulated NF-κB signaling and inflammatory pathways, we also analyzed inflammatory cytokine profiles and observed a significant increase in pro-inflammatory cytokines in the BM of Myd88 L262P mice compared to WT mice. Through single-cell RNA sequencing of hematopoietic cells (cKit+ enriched BM cells), we found that Myd88 L262P expression alters the expansion of distinct hematopoietic cell clusters, especially immature and mature neutrophil populations, and enrichment of innate immune and inflammatory pathways in HSPCs. We next determined whether the fitness advantage of MYD88 mutant HSPCs is due to NF-kB activation. For this, we performed a competitive BM transplantation using Myd88 L262P BM cells and then treated the mice with a dual IRAK1/4 inhibitor (NCGC-1481) for 6 weeks. Treatment with the IRAK1/4 inhibitor suppressed NF-kB activation in vitro and reversed the competitive fitness advantage of MYD88 L262P BM cells in vivo. In summary, we demonstrate that MYD88 gain-of-function mutations contribute to CH by inducing innate immune pathways in HSPCs and inflammatory cytokine expression. These findings offer new perspectives on the functional impact of genetic variants on HSPC fitness and the relevance of previously under reported CH mutations.

Background: Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. Methods: The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. Results: EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. Conclusions: Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

Background. Lomonitinib is a highly potent and selective pan-FLT3/ IRAK4 inhibitor that targets clinically relevant FLT3 mutations as well as IRAK4, a putative escape pathway for FLT3-driven AML. The relative selectivity of Lomonitinib compared to other FLT3 inhibitors was derived from a novel in silico modeling approach that avoided multiple alternative targets commonly inhibited with commercially approved agents. In vitro studies with FLT3-ITD cell lines demonstrated potent inhibition and differential decrease of FLT3 protein expression compared to gilteritinib. Multiple in vivo studies with both xenograft and syngeneic immune competent murine models demonstrated that lomonitinib has superior efficacy to gilteritinib in ITD and gatekeeper mutation-dependent disease and demonstrated synergistic efficacy when administered in combination with Bcl-2 or menin inhibitors1. Unlike gilteritinib, lomonitinib had minimal toxicity observed in pre-clinical rodent and dog toxicology studies at exposures well beyond the anticipated therapeutic exposure. A first-in-human clinical trial of lomonitinib in healthy volunteers was initiated in August 2023. Application of healthy volunteer studies with agents having a broad therapeutic index such as lomonitinib offer the opportunity to enter a subsequent AML patient clinical trial at a clinically effective dose. Additionally, these studies provide better prediction of tolerability in AML patients. We bring forward this new model of myeloid leukemia drug development for targeted therapies. Methods. We conducted a Phase 1, single-center, prospective, randomized, double-blind placebo-controlled study of lomonitinib administered orally to healthy adult participants (NCT06399315). The primary objective was pharmacokinetics (PK), the secondary objective was safety, and an exploratory objective was pharmacodynamics (FLT3 target engagement). This analysis focuses on the 3 MAD cohorts and a SAD cohort investigating drug-drug interaction (DDI) with itraconazole, a strong CYP3A4 inhibitor. Results. Due to the extended half-life and safety of lomonintib demonstrated in the Phase 1 SAD study2, we utilized a loading strategy (50 mg or 100 mg Day 1) to rapidly reach steady state (Css) to enable immediate therapeutic effect (versus delayed steady state exposures as observed with other FLT3 inhibitors). This was followed by a maintenance dose of 10 mg or 20 mg QD on Days 2-7. A total of 24 subjects were enrolled in the Phase 1 MAD study of lomonitinib. Six subjects dosed with 50 mg on Day 1 and 10 mg on Days 2-7, 6 subjects dosed with 50 mg Day 1 and 10 mg Days 2-7 in the presence of a proton pump inhibitor (PPI), and 6 subjects dosed with 100 mg on Day 1 and 20 mg on Days 2-7. Six subjects across 3 cohorts were dosed with placebo on Days 1-7. To provide a preliminary estimate of the effect of CYP3A4 inhibitors on the exposure level of lomonitinib, 6 subjects were dosed with 50 mg of lomonitinib on Day 1 combined with itraconazole (given BID on day -4 and QD on day -3 through day 1) as part of the SAD study. Lomonitinib was well tolerated with no treatment-related safety signals reported in any MAD or SAD cohort. Pharmacokinetic (PK) data from the MAD cohort 1 confirmed that the loading dose of 50 mg on Day 1 with 10 mg QD maintenance achieved steady state exposures by Day 4. The exposure of lomonitinib was minimally affected by the PPI and no significant influence on PK was observed with itraconazole. Consistent with studies in human cells in vitro, oral administration of lomonitinib in healthy human subjects demonstrated target engagement of FLT3-ITD in an ex vivo plasma inhibition assay at doses of ≥ 10 mg, which is anticipated to be in the therapeutic dose range based on preclinical data. Conclusion. Lomonitinib demonstrates favorable PK and safety profiles with dose-proportional increases in systemic exposure, FLT3 target engagement, and no treatment-related adverse events. Furthermore, little PK interaction was observed with protein pump or CYP3A4 inhibitors. The safety profile of lomonitinib enables rapid FLT3 engagement by using a loading dose (5-fold higher than the maintenance dose) which is not possible with other long half-life FLT3 inhibitors with less favorable therapeutic indices. A phase 1B study in R/R AML with mutated FLT3 is underway in Australia as well as in the US in collaboration with The Leukemia & Lymphoma Society Beat AMLâ. Sharpe, C., ASH 2023Byrd, JC, EHA 2024

Introduction: Inhibiting B-cell receptor (BCR) signaling with Bruton's tyrosine kinase (BTK) inhibitors in diffuse large B cell lymphoma (DLBCL) has benefited a subset of patients. The activated B-cell (ABC) subtype of DLBCL is critically dependent upon chronically active BCR signaling that promotes the assembly of the CBM adapter complex (CARD11, BCL10, and MALT1). The CBM complex enables MALT1-dependent recruitment of additional signaling proteins and unleashes MALT1 protease activity, engaging the downstream NF-ΚΒ pathway. While MALT1 promotes proper lymphocyte activation and development, it is also a significant contributor to the survival of aggressive lymphomas and other cancers. MALT1 is a promising target for chronic lymphocytic leukemia (CLL) in patients developing resistance to BTK inhibitors. Within the CBM complex, MALT1 provides both molecular scaffolding and protease activity. The scaffolding function of MALT1 provides early signaling events leading to NF-κB activation, while the protease activity of MALT1 serves to amplify and prolong this effect, and targeting both functions is optimal for an effective inhibitor. However, many existing MALT1 allosteric inhibitors only inhibit a single function through protease inhibition. Therefore, we describe here ZE66-0205, a novel, oral bioavailable MALT1 degrader with potent in vitro and in vivo activity. Results: Using CADD approaches and available crystal structures of the MALT1 protease we developed a highly active and selective MALT1 degrader, confirmed by proteomics studies in hPBMC cells. In vitro experiments with ZE66-0205 demonstrate inhibition of paracaspase activity in TMD8 cells (IC50 38 nM); inhibition of MALT1 dependent IL2 release in Jurkat cells (EC50 9 nM); and degradation of MALT1 protein in hPBMC cells (DC50 3 nM). In vivo PK experiments with ZE66-0205 demonstrated acceptable bioavailability, 20-25% in mice and 20-25% in dogs. To evaluate the MALT1 degrader ZE66-0205, the ABC-DLBCL cell line OCI-Ly3 (CARD11, MyD88 mutant) was treated with DMSO or ZE66-0205 (50, 500, 5000 nM) for 48 hrs. Immunoblots showed complete MALT1 degradation at all doses. Additionally, a dose dependent increase in full length RelB and CYLD was observed. Similar results were achieved in the TMD8 cell line (CD79b, MyD88 mutant). Interestingly, TMD8 cell lines CRISPR-edited to exclusively express BTK mutations seen with non-covalent (pirtobrutinib) T474I or covalent (acalabrutinib or zanubrutinib) C481S BTK inhibitors responded similarly to ZE66-0205. Furthermore, treatment of OCI-Ly3 at 500 nM ZE66-0205 led to >50% reduction of viable cells. To examine the effectiveness of ZE66-0205 in primary CLL B-cells, cells were treated with 1 or 10 μM for 48 hrs. and immunoblot showed complete degradation of MALT1 in comparison to DMSO treated control. Treatment with 10 μM of ZE66-0205 resulted in an 87% reduction (+19.7%) in viability versus DMSO control (n=3). To assess the efficacy of ZE66-0205 in degrading MALT1 in vivo, we utilized both wildtype and human celebron mutated mice (hu-CRBN, PubMed:30064974). The hu-CRBN mice are necessary to elicit any immunomodulatory function of immunomodulatory drug (IMiD) based degrader compounds. Mice were dosed with vehicle,10 mg/kg or 50 mg/kg ZE66-0205. A subset of mice (n=4 per group) received the drug orally for 4 days after which the mice were euthanized for pharmacodynamic analysis. Immunoblots in spleen lysates showed dose dependent degradation of MALT1 in the hu-CRBN mice. To assess survival efficacy of ZE66-0205 we used the OCI-Ly3 cell line disseminated xenograft model. The allosteric MALT1 inhibitor MLT-985 was used as a positive control. NCG mice were engrafted with OCI-Ly3 cells via the tail vein and mice received daily oral gavage of vehicle (n=11), 10 mg/kg ZE66-0205 (n=13) or 30 mg/kg MLT-985 (n=13) starting day 13 post-engraftment. Median survival was 37, 48, and 41 days, respectively. Analysis via Cox's proportional-hazards model shows superiority for the 10 mg/kg ZE66-0205 in comparison to vehicle group (p= 0.033). Conclusion: We describe a novel, orally bioavailable potent MALT1 degrader compound with good PK properties and in vivo efficacy. ZE66-0205 leads to on target degradation of CBM target proteins and represents a promising new treatment strategy for DLBCL and other B-cell malignancies. Future clinical development of ZE66-0205 is warranted and ongoing.

Abstract ID 93605Poster Board 428Background: Doxorubicin (DOX) is an anthracycline used in the treatment of various malignancies, including breast cancer and certain leukemias, but its use is limited by a debilitating cardiotoxicity. We previously reported that the uptake of DOX into cardiomyocytes is mediated by the organic cation transporter OCT3, and that inhibition of this mechanism ameliorates cardiotoxicity without affecting antitumor activity (Huang et al, 2021). Since OCT3 inhibition is not associated with altered plasma levels of DOX, alternative cardiac biomarkers of OCT3 are needed to optimize dosing strategies of OCT3 inhibitors used with DOX. We hypothesize that meta-iodobenzylguanidine (mIBG), an analog of norepinephrine used to image neuroendocrine tumors, can serve this purpose as it is a known substrate of OCT3 that accumulates in cardiac tissue.Methods: Pharmacokinetic studies were performed in wild-type mice and age- and sex-matched mice with a genetic deficiency of OCT3 or the related transporters OCT1 and OCT2 (OCT1/2), MATE1, or OCT1/2 and MATE1 (OCT1/2/MATE1). Non-radiolabeled mIBG was injected i.v. as a bolus dose at 15 mg/kg. Plasma and heart samples were collected at various time points (up to 4 hours), and pharmacokinetic parameters were calculated using Phoenix WinNonlin. In separate experiments, mice received 0.5 mCi of iodine-123 labeled mIBG for single-photon emission computerized tomography (SPECT-CT) scanning. Images from the axial, coronal, and sagittal perspectives were gathered 30 min after injection, and signals were quantified in cardiac tissue of wild-type and OCT3-deficient mice.Results: While the observed AUC of unlabeled mIBG in plasma was similar between wild-type mice and transporter-deficient animals, the levels of mIBG in hearts of OCT3-deficient mice were statistically significantly reduced compared to wild-type mice with mean heart-to-plasma ratios of 3.5 ± 0.598 and 38 ± 8.05, respectively (P<0.001). Levels of mIBG in the hearts of mice deficient in OCT1, OCT2, and/or MATE1 were either unchanged or slightly increased compared to results obtained in wild-type mice. In the SPECT-CT images, wild-type mice showed a higher average cardiac accumulation of 23.3E+05 Bq/mL of mIBG compared to OCT3-deficient mice with an average of 6.13E+05 Bq/mL (P<0.0001). There was not a significant difference in the uptake in the liver of these animals.Discussion: These findings confirm that radiolabeled mIBG can be utilized in conjunction with SPECT-CT scans as a non-invasive cardiac biomarker of OCT3 function, and that deficiency in OCT3 results in a stark decrease in signal. We are currently planning a high-throughput screen to identify novel small-molecule inhibitors of OCT3 that can be tested for OCT3-modulatory properties in vivo using a newly developed transgenic mouse model with cardiomyocyte-specific expression of human OCT3. By repeating these experiments with and without OCT3 inhibitors, we hope to observe a similar drastic decrease in mIBG signal in cardiac tissue upon OCT3 inhibition. It is expected that the proposed strategy can ultimately be translated to patients with cancer requiring treatment with DOX-based regimens to ameliorate cardiotoxicity.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

Mutations in protein tyrosine phosphatase non-receptor type 11 ( PTPN11 ) have been considered late acquired mutations in acute myeloid leukemia (AML) development. To interrogate the ontogeny of PTPN11 mutations, we utilized single-cell DNA sequencing and identified that PTPN11 mutations can occur as initiating events in some AML patients when accompanied by strong oncogenic drivers, commonly NPM1 mutations. The co-driver role of PTPN11 mutations was confirmed in a novel murine model that exhibits an AML phenotype with early expansion of a diverse set of variably differentiated myeloid cells that engrafted into immunodeficient and immunocompetent mice. This immune diversity was reconstituted from early precursor cells when engrafted into immunodeficient mice. Moreover, immune diversity was also observed in the blast component of patient samples with NPM1 and PTPN11 mutations, providing novel antigen targets for immune based approaches in this subset of AML that is resistant to multiple targeted therapies.

Adult patients with acute myeloid leukemia (AML) have a proportionate increase in mortality from their disease and therapy as age increases. In great part, this mortality is due to disease relapse that occurs at a higher rate irrespective of genetic assigned risk. While intensification of therapy in patients with favorable genomics such as core-binding-factor AML (CBF-AML) has yielded a high proportion of long-term remission and potential cure, this benefit has been less frequently observed in patients with AML age 60 and older despite receipt of similar therapy. Moreover, even less benefit has been observed in patients with unfavorable genomics such when bearing a complex karyotype (karyotype with ≥ 3 chromosome abnormalities). Reasons for this unfavorable outcome among older patients is unknown. We hypothesized that tumor-induced aging in cells of bone marrow microenvironment, specifically, in bone marrow mesenchymal stromal cells (BMSCs) plays a significant role in AML survival and progression in older compared to younger patients and that the difference in prognosis may depend on specific aging induced changes in the BMSCs. We expanded BMSCs under physiologic hypoxic conditions (91% nitrogen, 4% oxygen and 5% CO 2) to mimic the bone marrow microenvironment, isolated DNA and RNA from early passaged cells and performed methylation analysis using 850k Illumina DNAmehtylation arrays and gene expression analysis using mRNAseq. We used the Horvath Skin and Blood clock to calculate epigenetic age (DNA methylation age). We determined differentially expressed genes with increasing epigenetic or chronological age in the different AML genetic risk groups (n=9 CBF-AML, n=11 CK-AML and n=7 Normal donor BMSC) as well as in older (>60) and younger (<60) AML-BMSCs, and performed Gene Set Enrichment Analysis (GSEA) for the Hallmarks as well as aging and senescence related genesets (n=33 genesets). We found that BMSCs derived from AML bone marrow cells are more age accelerated than normal donor BMSCs. Our GSEA analysis revealed enrichment for age-associated gene networks that reflected hallmarks of aging, including mitochondrial dysfunction, inflammation, and cellular senescence. Specifically, we observed a positive enrichment for the SenMayo gene signature ( Saul et al. 2022, Nature Communications) and a negative enrichment of proliferation and mitotic gene sets with increasing epigenetic and chronological age (padj<0.05). Gene sets that are related to inhibition of adipogenesis were positively enriched in the CBF-AML BMSCs compared to the CK-AML BMSCs. Our integrated analysis revealed novel observations regarding the cellular changes that occur in the AML stromal microenvironment with increasing age. Most interestingly, our findings suggest that AML derived BMSCs have increased epigenetic age compared to normal donor BMSCs. Additional experiments are ongoing to functionally validate the findings predicted by the enrichment analysis. Our study is the first to suggest an increased epigenetic age in the stromal bone marrow microenvironment of older AML patients.

Cellular senescence is a durable cell cycle arrest as a result of the finite proliferative capacity of cells. Senescence responds to both intrinsic and extrinsic cellular stresses, such as aging, mitochondrial dysfunction, irradiation, and chemotherapy. Here, we report on the use of mass cytometry (MC) to analyze multiple model systems and demonstrate MC as a platform for senescence analysis at the single-cell level. We demonstrate changes to p16 expression, cell cycling fraction, and histone tail modifications in several established senescent model systems and using isolated human T cells. In bone marrow mesenchymal stromal cells (BMSCs), we show increased p16 expression with subsequent passage as well as a reduction in cycling cells and open chromatin marks. In WI-38 cells, we demonstrate increased p16 expression with both culture-induced senescence and oxidative stress-induced senescence (OSIS). We also use Wanderlust, a trajectory analysis tool, to demonstrate how p16 expression changes with histone tail modifications and cell cycle proteins. Finally, we demonstrate that repetitive stimulation of human T cells with CD3/CD28 beads induces an exhausted phenotype with increased p16 expression. This p16-expressing population exhibited higher expression of exhaustion markers such as EOMES and TOX. This work demonstrates that MC is a useful platform for studying senescence at a single-cell protein level, and is capable of measuring multiple markers of senescence at once with high confidence, thereby improving our understanding of senescent pathways.

PDF file - 82K, Supplementary Table 1: Fold changes of NF-κB target genes in CLL: CLL cells (n=12) were treated with 300 nM CFZ (1 hr), 1.7 M CpG (4 hr) or 500 ng/ml CD40L (4 hr). RNA was extracted at 8 hr from CFZ treated samples and mRNA expression was analyzed using real-time RT-PCR. Cells highlighted in light grey have estimated fold changes > 2 and were significant at the (unadjusted) α=0.05 level of significance. Cells highlighted in dark grey are significant at the α=0.05 level, but have estimated fold changes < 2. Supplementary Figure 1: Selection of 697 cells to generate p53DN cells: Jeko, MEC-1 and 697 cells (n=3) were exposed to 0, 300 nM CFZ for 1 hr and 5 Gy gamma irradiation. (S1A) Viability was assessed by MTS assay at 48 hrs and results were calculated relative to time-matched samples untreated samples. (S1B) Whole cell lysates and RNA were collected at 8 hr and were immunoblotted for indicated proteins using GAPDH as a loading control. (S1C) Changes in transcription of p53 and subsequent targets were assessed by real-time RT-PCR.

This includes additional material for the paper including 1) Table 1. Microarray results for genes of interest; 2) Figure s1 showing decrease in IKZF1 and IKZF3 by lenalidomide; and Figure S2. Lenalidomide, IL-21, and the combination of lenalidomide + IL-21 induce different transcriptional signatures