Myelodysplastic Syndromes Samples Research Articles

Abstract 6744: Activating anti-MDS immunity with polarized dendritic cell vaccines

Abstract Less than 50% of patients with higher-risk myelodysplastic syndromes (MDS) show clinical response to standard-of-care hypomethylating (HMA) therapy. Significant enthusiasm existed for immunotherapy/HMA combinations for these patients, but studies have failed to deliver on the initial promise. We have demonstrated that patients with MDS have a deficiency in antigen-presenting cells, called CD141Hi dendritic cells (DC). DC-deficient MDS patients have reduced response to HMAs, worse overall survival, and show less response to cancer vaccination. We hypothesized that restoration of this population using a novel DC vaccination approach - alpha-type-1 polarized dendritic cells (αDC1) - might compensate for this deficiency and restore immunity. To generate αDC1s, we collected monocytes from MDS patients and healthy donors (HD) and cultured them for 6 days in vitro in the presence of recombinant human GM-CSF and IL-4 followed by an additional 18-24 hours in the presence of αDC1 maturation factors (poly-I:C/IFNα/IFNγ/TNFα/IL-1β). Overall, we attempted to generate αDC1s from 15 MDS samples; 13/15 exhibited intermediate to very high-risk disease and 10/15 are currently receiving HMA therapy. We successfully cultured cells from 8/15 samples (53%). Of the 8 MDS samples, 5 were collected from patients receiving HMA therapy (62.5%). We were unable to successfully culture αDC1s from the other 5 patients receiving HMA therapy, and thus the effect of HMA therapy on aDC1 generation remains unclear. To test the function of MDS αDC1s, we focused on two signals necessary to activate immune responses: 1) the expression of co-stimulatory molecules and 2) the production of IL-12p70. Flow cytometric analysis on αDC1s and immature DCs from MDS samples revealed intra-patient variation in the fold-change surface expression of CD40/80/83/86 and HLA-DR proteins. Additionally, there was a trend towards decreased up-regulation of CD80 and CD86 in MDS specimens compared to HD. To assess production of IL-12p70, we stimulated immature DCs and αDC1s with CD40L and performed ELISA analysis on the media supernatant. We found that 3 out of 4 samples could produce IL-12p70 (range 27 - 5,480 pg/ml). To date, our results suggest that generation of MDS patient-derived αDC1 cells is feasible and additional work is being performed to fully characterize their ability to activate anti-MDS immune responses. Since αDC1s have an established safety profile in patients with solid tumors, we anticipate that we will be able to rapidly translate our results to the clinic. Citation Format: Daniel Alvarado, Jennifer Peresie, Adam Brinkman, Cheryl Allen, Weijian Jiang, Pawel Kalinski, Tiffany Tanaka, Elizabeth Griffiths, Michael Nemeth. Activating anti-MDS immunity with polarized dendritic cell vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6744.

Abstract 4628: Validation of an automated, scalable comprehensive genomic profiling assay for hematologic malignancies

Abstract Optimal diagnosis, prognosis, and treatment selection for hematologic malignancies requires assessment of somatic mutations across a subset of clinically relevant genes. Here we present the analytical validation results of an expanded targeted next generation sequencing (NGS) panel of 141 clinically relevant genes for myeloid and lymphoid malignancies. Based on review of clinical guidelines, 141 genes including 79 myeloid and 84 lymphoid associated genes were selected. The expanded hematological panel interrogates all coding exons of the 141 genes to detect single nucleotide variants (SNVs) and insertions/deletions (indels). The panel also identifies copy number aberrations (CNAs) in 16 genes, FLT3 internal tandem duplicates (ITDs), select non-coding pathogenic variants, and patient sex. The custom hybrid capture-based assay utilizes genomic libraries created from 250 ng gDNA extracted from peripheral blood, bone marrow, or cell suspensions, followed by sequencing on Illumina® instruments. Concordance studies were performed on clinical samples previously assessed using an orthogonal NGS-based laboratory developed test for SNVs/indels/FLT3-ITDs or digital multiplexed ligation-dependent probe amplification for CNAs. In total, 308 patient samples were evaluated and included 72 (23.4%) acute myeloid leukemia and 58 (18.8%) myelodysplastic syndromes samples among other indications, of which 240 (77.9%) were bone marrow specimens. Analysis of concordance demonstrated a positive percent agreement (PPA) of 99.7% for SNVs (750/752), 99.5% for indels <25bp (206/207), and 95.7% for indels ≥ 25bp (22/23). Variants were from 45 genes including TP53, NPM1, and IDH1/2. PPA was 100% for FLT3-ITDs (38/38; size range 18-300 bp) and sex (294/294). For CNAs, PPA was 96.4% (132/137) and negative percent agreement (NPA) was 98.3% (931/947). Assay precision was determined using three replicates of 15 clinical samples for both intra and inter-assay precision using multiple operators, instruments, and reagent lots. 100% concordance was observed for SNVs (36/36), indels (11/11), a 24 bp FLT3-ITD (1/1), CNAs (24/24), and sex (15/15). Analytical specificity was assessed using 5 replicates of NA12878 showing specificity >99.99% for SNVs/indels with variant allele frequency ≥ 3%, and >99.99% for CNAs with copy number ≤0.85 or ≥1.15. Dilution series to determine analytical sensitivity are in progress. This study highlights the analytical validation of the expanded NGS panel for the detection of clinically informative genomic alterations in hematologic malignancies. Results from this validation study, when complete, will include at least 576 samples plus orthogonal testing. These data describe the performance of the assay to enable a comprehensive evaluation of genomic alterations using a single sample, further facilitating the use of broad NGS assays in patients with hematologic malignancies. Citation Format: Grant Hogg, Tong Liu, Helen Cao, Adib Shafi, Ashraf Shabaneh, Jon Williams, John Howitt, Amanda Williamson, Rachel Dango, Xiaojun Guan, Heidi Hoffmann, Michael Mooney, John Pruitt, Scott Parker, Henry Dong, Stan Letovsky, Li Cai, Eric A. Severson, Shakti H. Ramkissoon, Anjen Chenn, Marcia Eisenberg, Brian Caveney, Eyad Almasri, Taylor J. Jensen. Validation of an automated, scalable comprehensive genomic profiling assay for hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4628.

Single-cell Multiomics Analysis of Myelodysplastic Syndromes and Clinical Response to Hypomethylating Therapy.

MDS are myeloid clonal hemopathies with a low 5-year survival rate, and approximately half of the cases do not respond to standard HMA therapy. Our innovative single-cell multiomics approach offers valuable biological insights and potential biomarkers associated with the demethylating agent efficacy. It also identifies vulnerabilities that can be targeted using personalized combinations of small drugs and antibodies.

Reduced Subclone Diversity in Clonal Cytopenia of Undetermined Significance Compared to Myelodysplastic Syndrome

Introduction: Clonal cytopenias of undetermined significance (CCUS) and myelodysplastic syndromes (MDS) represent a disease continuum that are distinguished by the presence of dysplasia in >10% of blood cells. However, morphologic dysplasia is subject to high inter-observer variability, suggesting that dysplasia may not be ideal to differentiate CCUS from MDS. Defining the clonal architecture of samples from cytopenic patients may provide a more accurate objective measure of disease status than dysplasia. We hypothesize that the number and size of subclones is reduced in patients with CCUS compared to MDS, suggesting that increased subclonal diversity is a hallmark of higher-risk disease. Methods: We performed whole genome sequencing with higher exome coverage (eWGS) on bone marrow (n=58) or peripheral blood (n=4) and paired normal DNA from baseline banked samples from patients with CCUS (n=13), MDS (n=29; IPSS-R very low/low [3], intermediate [5], high/very high [19], no score [2]), and secondary AML (sAML) (n=20), including 32 previously reported patients, to define clonal architecture. Putative somatic variants, including 76 genes that are recurrently mutated in myeloid neoplasms, were validated in hematopoietic and paired normal samples, and available serial samples (n=23), using orthogonal target enrichment sequencing platforms achieving higher coverage (~600-1000x). Clonality of validated somatic mutations was defined using SciClone. Results: The absolute neutrophil count, hemoglobin, and platelet counts trended lower in MDS vs CCUS patients (median, 1.3 vs 2 K/ul [ns]; 9.3 vs 11.35 g/dL [p<0.01]; 63 vs 109.5 K/uL [ns], respectively). The ages and median number of total validated somatic mutations identified by eWGS at baseline was similar for CCUS (n=428), MDS (n=300), and sAML patients (n=420.5). We first defined the founding clone (i.e., the dominant clone with the highest median variant allele frequency [VAF]) and subclones. While the number of total clones per sample was not different between CCUS, MDS, and sAML (median, n=3 for each), the median VAF of all the mutations present in the founding clone (i.e., measure of the maximal molecular disease burden) was significantly lower in CCUS (21.3%) compared to MDS (39.2%, p<0.05) and sAML samples (45.2%, p<0.001), but MDS and sAML were not different (p=0.2) (Figure 1A). In contrast, there was no significant difference in the median VAF of subclones in CCUS and MDS samples (13.4% vs 16.8%), which were both lower than sAML (25.3%, p<= 0.01). However, the proportion of patients with no subclones (i.e., only the founding clone detected) was higher for CCUS (5/13 [38.5%]) compared to MDS and sAML (4/29 [13.8%] and 0/20 [0%], respectively, p 0.0006), suggesting that CCUS samples have reduced subclonal diversity compared to MDS. Consistent with this, genes commonly mutated in subclones occurred less frequently in CCUS vs MDS samples, including activated signaling (0/13 vs 6/29) and transcription factor genes (3/13 vs 9/29, respectively). Nine CCUS patients had serial samples sequenced (median 2 serial samples, range 1-5) at a median follow-up of 539 days (range, 84-4066 days). In CCUS patients with serial samples, there was minimal change in the founding clone (0.16%, range -3.7-23.9%) or the subclone median VAFs (0.2%, range -22.4-22.2%), with only 2 patients having a founding clone or subclone VAF increase of >5%. UPN 529198 had the acquisition of a new SRSF2(P95H)-mutant subclone 4066 days after initial banking but remained with stable cytopenias and no MDS (Figure 1B). One CCUS patient developed MDS 70 days after banking with a blast count that increased from 2% to 15% but had a negligible change in the founding clone VAF (2.8% increase). However, 3 subclones did increase in size (max VAF increase of 6.1%). In summary, while the total number of mutations is similar between CCUS, MDS and sAML samples, the clonal architecture varies across diseases. CCUS has a lower total molecular disease burden and a lower proportion of patients with a subclone compared to MDS patients in our cohort. The data suggest that defining clonal architecture and incorporating subclonal complexity in the evaluation of cytopenic patients could provide an objective measure to characterize and monitor disease burden rather than relying on the presence or absence of dysplasia, especially when considering the diagnosis of CCUS and lower-risk MDS.

Inhibition of Toll-like Receptor 4 (TLR4) Promotes Erythroid and Myeloid Differentiation in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Introduction: The hallmark of Myelodysplastic Syndrome (MDS) is a block in differentiation of hematopoietic precursors resulting in single or multilineage cytopenias. Evaluation of regulatory pathways that are inappropriately overactive and lead to a blockade in erythroid and myeloid differentiation is an important process in determining future therapeutic targets for the treatment of MDS. The Toll-like receptor (TLR) pathways play an important role in the innate immune response and are also highly expressed on hematopoietic stem cells. Aberrant overactivation of these pathways can stimulate inflammation and cytokine production in the marrow microenvironment. Also, while the overactivation of interleukin-1 receptor-associated kinase 4 (IRAK4), tumor necrosis factor receptor associated factor 6 (TRAF6) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways has been seen in Myelodysplastic Syndrome and Acute Myeloid Leukemia (AML), the upstream signals that drive their activation are not fully identified. Results: We investigated the expression of all TLRs in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that TLR6 and TLR4 were the most significantly overexpressed receptors in MDS samples (FDR<0.001). Furthermore, we observed that the TLR4 cofactor lymphocyte antigen 96 (LY96), also known as myeloid differentiation factor 2 (MD-2), was also overexpressed in MDS samples when compared to controls. Furthermore, the S100A8 and S100A9 proteins are ligands for TLR4 and were found to be positively associated with severity of anemia in the cohort of MDS samples. These data provided the rationale for determining the functional role of the TLR4 pathway in MDS/AML models. To obtain a potent and specific antibody against TLR4, we inoculated rabbits with recombinant human TLR4 and obtained high affinity antibodies that neutralized TLR4 function at low nano-molar range of doses. The TLR4 polyclonal antibody was able to inhibit lipopolysaccharide (LPS)-induced NF-κb activation in leukemic THP-1 cells. The TLR4 antibody also led to a decrease in proliferative phosphorylated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase in leukemic cells. We then treated samples from MDS/AML patients (n=14) with the TLR4 antibody (1 nM dosing) and assessed for erythroid and myeloid growth and differentiation. Fluorescence activated cell sorting (FACS) analysis for erythroid differentiation showed increased expression of either CD71, CD235a or both after treatment with the TLR4 antibody. FACS analysis for myeloid differentiation showed increased expression of either CD11b, CD14 or both after treatment with the TLR4 antibody. This effect was more pronounced in the intermediate/high risk group than in the low-risk group of patients. Conclusion: Inhibition of the TLR4 pathway with a potent TLR4 antibody promotes erythroid and myeloid maturation and relieves the differentiation block on these lineages in MDS patient samples in the in vitro setting. Our preclinical data supports further evaluation of TLR4 inhibition as a therapeutic target in patients with MDS, as anemia and neutropenia are hallmarks of the disease.

Novel Clinically Useful Inhibitor of Mediator Complex, RVU120, Relives Differentiation Block in MDS/AML

Background: Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemias (AML) are characterized by a block in hematopoietic differentiation that can often be traced to aberrant stage-specific transcription. The kinase module of the mediator complex, consisting of CDK8, its paralog CDK19, Cyclin C, MED12, and MED13, have been identified as a molecular switch that regulates gene expression. A variety of cancers have exploited this mechanism to maintain stemness and an undifferentiated state. RVU120 is a first-in-class, specific, and selective inhibitor of CDK8/19, currently in Phase Ib clinical trial in patients with AML or High-Risk MDS (HR-MDS) (NCT04021368). RVU120 has been shown to stimulate erythroid differentiation in transformed CD34+ cord blood (CB) cells and in CD34+ cells from Diamond-Blackfan Anemia (DBA). To evaluate the further therapeutic potential of CDK8/19 inhibition, we confirmed aberrant expression of mediator complex components and erythroid-stimulating activity in a collection of CD34+ primary samples of AML and MDS. Results: We investigated the expression of mediator complex components in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that MED12, a critical component of the mediator complex, was significantly overexpressed in MDS samples from refractory anemia with excess blasts (RAEB), a higher risk subset of MDS(p=0.018) (Fig A). Furthermore, we observed that this overexpression of MED12 was associated with a higher rate of transformation to AML. To delve deeper, we examined the expression of MED12 in sorted leukemic stem cells from primary AML samples and found significantly elevated levels in Long Term Hematopoietic Stem Cells from AML samples with both normal karyotype and complex karyotypes compared to healthy controls. Next, we conducted an evaluation of the efficacy of the mediator kinase module inhibition in MDS-derived cell line (MDS-L). Inhibition of CDK8/19 with RVU120 led to increased erythroid differentiation in this model which confirmed our observations in transformed CD34+ CB cells in CB cells, these phenotypic changes were correlated with the inhibition of CDK8-dependent phosphorylation marker S726 on STAT5 and a profound increase in the expression of genes involved in erythroid commitment and hemoglobin metabolism. In order to confirm therapeutic potential of CDK8/CDK19 inhibition in MDS and AML, we performed colony-forming assay on primary samples of MDS and AML (n= 15). The treatment with RVU120 resulted in increased erythroid differentiation in a majority of samples, evident from an augmented number of colonies. Detailed analysis indicated changes in the expression of erythroid differentiation markers, including increased CD71 and Glycophorin A expression on colonies, as assessed by FACS analysis (Figure B, shows representative sample). Conclusion Our data highlight that inhibition of the overexpressed mediator complex proteins can alleviate the differentiation blocks seen in MDS/AML. These results provide a compelling rationale for further developing the CDK8/19 inhibitor RVU120 for transfusion- dependent MDS/AML patients.

Deregulation of HLA-Class I and Class II Genes in Chip, MDS, and Acute Leukemia and Similarity between Chip and MDS in HLA Profile

Introduction: Human leukocyte antigen (HLA) plays a major role in the interaction between the immune system and oncogenic process in various types of tumors. Emerging data suggests that the HLA system may have profound effects on response to immunotherapy and other types of therapy. Structural defects in HLA genes and variation in methylation have been reported to play a role in the expression of various HLA-I and HLA-II genes. Furthermore, an association between the expression of specific HLA-I or HLA-II genes and prognosis and outcome has been reported in some types of leukemia. Since HLA-I is expressed on all cells while HLA-II is expressed mainly in antigen presenting cells, we hypothesized that profiling the expression levels of both HLA-I and HLA-II in the bone marrow of patients with various myeloid neoplasms may provide information on the interaction between the microenvironment and the leukemic cell in these diseases. We used next generation sequencing to evaluate the expression level of HLA-I and HLA-II genes in myeloid neoplasms and compared these levels to normal bone marrow control. We used targeted next generation sequencing (NGS) for quantification of the RNA expression of 15 HLA-I and HLA-II genes. Methods: RNA was extracted from fresh bone marrow aspiration samples from 115 acute myeloid leukemia (AML), 196 clonal hematopoiesis of indeterminate potential (CHIP), 84 myeloproliferative neoplasm (MPN), 177 myelodysplastic syndrome (MDS), 40 acute lymphoblastic leukemia (ALL), and 279 normal bone marrow cases. RNA sequencing was performed using a targeted hybrid capture panel. The sequenced HLA-I genes were HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-H. The sequenced HLA-II genes were: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB5, and HLA-DRB6. Salmon v1.4.0 software was used for expression quantification (TPM). To evaluate the depth of the significance in variation of the expression profiles between diseases we tested machine learning algorithm (random forest) incorporating the expression of all 15 genes in blindly classifying diseases. Two thirds of samples were used for training the random forest algorithm and one third was used for testing. Results: Bone marrow samples with CHIP showed no significant difference from normal bone marrow in level of expression of HLA-I genes but showed significantly (P <0.001) lower level of most HLA-II markers (DMB, DQA1, DQB2, DRB1, and DRB5). The same pattern was observed in MDS samples. In contrast, AML showed significant (P < 0.001) increase in the expression of both HLA-1 genes (A, C, and F) and HLA-II genes (DQA1, DPA1, DQB1, DRB6, and DPB1). Bone marrow samples from ALL patients showed significantly (P< 0.001) higher expression levels in DRB1 and DQA1 as compared with normal bone marrow. MPN samples showed no statistically significant difference from normal BM in any of the HLA-I or HLA-II genes, after adjusting for multiple testing. Machine leaning algorithm poorly classified MDS and AML and suggests complex overlap between the two diseases in HLA response. In contrast, the machine learning algorithm showed significant difference between MPN, MDS, and AML, and ability to distinguish all MPN cases with AUC of 0.738 (95% CI : 0.546-0.930) in the testing set (see figure). Conclusions: These findings show that the bone marrow microenvironment immune profile in MDS and CHIP likely plays a major role in the disease. Furthermore, this data suggests that this immune microenvironment is critical in CHIP and perhaps that therapeutic approaches supporting this immune microenvironment may help in preventing CHIP from progressing to MDS. The data also supports that AML is a disease that completely overwhelms the HLA immune system. In contrast, MPN appears to evade the HLA system and perhaps activating this system may help in the therapy of MPN.

Enhancing Myeloid Cancer Genomic Profiling through Comprehensive Whole Genome Sequencing Using the Isabl Gxt Heme Analytical Platform

Introduction: Genomic profiling in myeloid cancers is vital for disease classification, risk stratification, and treatment decisions. Whole genome sequencing (WGS) captures a complete range of biomarkers, overcoming conventional diagnostic constraints of multiple assays (Duncavage et al, 2021). Duncavage et al's seminal work demonstrated WGS as an alternative and sensitive approach to cytogenetics. Due to emerging evidence, NCCN integrated WGS in their AML testing guidelines (v2.2023). Given the importance of WGS in myeloid cancers, we developed Isabl GxT Heme, an analytical platform, that uses unmatched WGS data as input, assesses all mutation types across the entire genome (structural variants (SVs) and copy number variations (CNVs), small mutations) and delivers an automated report of clinically relevant findings in accordance to latest clinical guidelines. Here we use the Duncavage dataset to analytically validate this WGS approach for the Isabl platform. Methods:Data for 263 samples from Duncavage et al were processed using Isabl GxT Heme analytical framework. The cohort included 175 AML, 81 myelodysplastic syndrome (MDS), and 7 other myeloid malignancies, with a median coverage of 50x (19x - 90x). 2 samples were excluded due to low coverage (<20x). Sensitivity analysis for Isabl GxT Heme and ChromoSeq (reference test; Duncavage et al., 2021) involved comparing against clinical panel sequencing for single nucleotide variations (SNVs) and indels, and against cytogenetics for SVs and CNVs (restricted to clonal events). Results:Isabl GxT analytical workflow returned results within one day from FASTQ generation (<19 hours). Isabl GxT Heme and the reference test detected 100% of recurrent SVs (41/41) and clonal CNVs (67/67) that were reported by cytogenetics. Isabl GxT Heme sensitivity for SNVs and indels was 93% (219/236) and 93% (109/117) for all events called by panel sequencing. Sensitivities for the reference test were 85% and 92%, respectively. A total of 850 calls from 261 samples from the reference test were evaluated next (41 SVs, 205 SNVs, 113 Indels, and 491 CNVs). Isabl GxT Heme identified 100% of SVs, while sensitivity for SNVs was 99.5% (204), and that for indels was 98% (111). Isabl GxT Heme called 97% (475) of CNVs with clonality >20%. Our sensitivity in calling FLT3 internal tandem duplication (ITD) as compared to polymerase-chain-reaction (PCR)-based assay was 84% (27/32) as compared to the reference test at 87% (28/32). Of the 46 genes from the latest guidelines for myeloid disease classification and risk stratification (guideline genes; WHO-2022, IPSSM-2022), 12 were not reported by the targeted analytical approach of the reference test and panel sequencing. A total of 32 guideline related small mutations in 29 samples (11%) were called by Isabl GxT Heme alone . In 13/29 samples, the called events were deemed oncogenic/likely oncogenic by OncoKB (Chakravarty et al. 2017). Isabl GxT Heme's allele-specific approach detects copy-neutral loss of heterozygosity (cnLOH), identifying 7 samples (3%) with bi-allelic TP53 loss due to a small mutation and cnLOH. Additionally, 9 focal CNVs (<5Mbp) were detected in 6 samples (2%). Partial tandem duplication eventsinKMT2A(MLL PTD) in 4 AML and 2 MDS samples were uniquely reported by Isabl GxT Heme. 9 SVs were reported affecting guideline genes (Table 1) . Cytogenetics and panel sequencing information was available for 94 samples in this cohort, enabling an assessment of the validity of Isabl GxT Heme. Additional myeloid-relevant driver events not detected by panel sequencing/cytogenetics were found in 27% of patients by Isabl GxT Heme and 12% by the reference test. Isabl GxT Heme called events in 30% of patients as compared to the reference test (Fig 1). All events missed by Isabl GxT Heme were small mutations detected by panel sequencing of which 69% were absent in the aligned BAM file (11/16) and 31% (5/16) had a median variant allele fraction of 5%. Conclusion:Isabl GxT Heme represents a myeloid cancer profiling solution that reports all clinically relevant biomarkers in AML and MDS inclusive of TP53 allelic state, MLL PTD, disease defining and risk stratifying biomarkers across mutation types. Isabl GxT Heme had comparable sensitivity to the reference test and additionally detected myeloid-relevant driver events in 27% of patients. This work demonstrates the validity and added benefit of Isabl GxT Heme in characterizing myeloid cancers.

Sting-Mediated Interferon Signaling Exerts Potent Antileukemic Effects in High-Risk Myeloid and Monocytic Malignancies

Clinical outcomes of patients (pts) with high-risk (HR) myeloid neoplasms remain dismal despite available therapies. Interferons (IFN) regulate hematopoietic stem-progenitor cell (HSPC) differentiation, proliferation and self-renewal capabilities and preferentially impair clonal HSPC fitness. To evaluate if myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) HSPCs activate IFN responses we performed RNA sequencing in bone marrow (BM) CD34 + cells obtained from pts with MDS (n=58), CMML (n=27) and age-matched healthy donors (HDs, n=17). This analysis revealed significant deregulation of IFN pathway genes in HSPCs from pts with CMML and to a lesser extent in those of pts with MDS. Activation of the stimulator of interferon genes (STING) can inhibit stem-cell function and induce myeloid and monocytic cell death via IRF3-dependent and independent mechanisms. Therefore, we hypothesized that STING pathway activation might have therapeutic potential in high risk (HR) myeloid and monocytic (MyMo) neoplasms. In vitro treatment of monocytic MOLM13 and THP1 cell lines with the novel synthetic STING agonist dazostingag resulted in induction of IRF3 phosphorylation, caspase 3 cleavage and apoptosis. Dazostingag was also able to induce enhanced cell death in multiple myeloid cell line cultures. Given the ability of dazostingag to induce apoptosis of monocytic cell lines, we sought to evaluate the activity of monocyte-directed STING activation using TAK-500, an IgG1 anti-CCR2 antibody linked to dazostingag as a STING agonist payload. To do so, we first analyzed the expression of the monocyte-specific cysteine-cysteine chemokine receptor 2 (CCR2) in primary pts samples and cell lines. Both CCR2 RNA and protein expression levels were higher in CMML compared to HDs or MDS. In accordance with this finding, monocytic cell lines (MOLM13, THP1) had significantly higher CCR2 expression compared to non-monocytic cell lines (SKM1, HL60, U937, KG1). Treatment of MOLM13 and THP1 cell lines with TAK-500 induced strong IRF3 phosphorylation, caspase 3 cleavage and apoptosis. In vitro treatment of BM mononuclear cells (MNCs) from pts with MDS (n=10), CMML (n=11) or AML (n=8) in co-culture with mesenchymal stromal cells with TAK-500 (0.5 mM) or dazostingag (1 mM) resulted in increased cell death of MNCs from pts with CMML and AML. TAK-500 (0.5 mM) significantly depleted CCR2 + BM MNCs from all disease groups but did not affect CCR2 + cells from HDs. Next, to evaluate whether STING activation impairs repopulation potential of neoplastic HSPCs we performed colony-formation studies in CD34 + BM cells from pts with CMML (n=8), MDS (n=3) and AML (n=4). Treatment with TAK-500 (0.5mM) and dazostingag (1mM) resulted in significantly impaired myeloid colony-formation in all disease groups without significantly impairing erythroid colony-formation in MDS or CMML samples. Taken together, these data suggests that STING activation might induce aberrant myelomonocytic cell death and deplete aberrant HSPCs in HR-MyMo neoplasms. To evaluate the in vivo efficacy of TAK-500 and dazostingag, we established xenograft models by inoculating NOG-IL15 transgenic mice with MOLM13 cells. Treatment with TAK-500 and dazostingag resulted in reduction of tumor burden evaluated by bioluminescence with no observed toxicities. Administration of weekly doses of TAK-500 induced enhanced and sustained tumor burden suppression. To determine the in vivo effect of STING activation in an immune competent model of CMML, we then evaluated the effects of TAK-500 and dazostingag in Vav-KDM6B/Tet2 f/f/Vav-Cre mice, a previously reported (Wei et al Leukemia 2022) animal model with CMML phenotype. Treatment with TAK-500 led to enhanced apoptosis of Ccr2 + BM cells in these mice. Moreover, the number of colonies formed by lineage negative/Sca1 positive/cKit positive BM progenitor (LSK) cells isolated from TAK-500 treated mice was significantly lower than in vehicle treated control mice, suggesting a negative regulation of repopulating capacity of Tet2 deficient/ KDM6B overexpressing BM HSPCs by TAK-500. Evaluation of the efficacy of TAK-500 and dazostingag in patient-derived xenograft models of primary CMML samples is planned. Taken together, our data suggests that CCR2-directed STING activation might have therapeutic potential in HR-MyMo neoplasms by depleting CCR2 + cells and impairing clonal HSPC repopulation potential.

Molecular Taxonomy of Myelodysplastic Syndromes and Its Clinical Implications

Background Recurrent genetic abnormalities are responsible for the pathogenesis of myelodysplastic syndromes (MDS) and their biologic heterogeneity, however current classifications of MDS are still largely based on morphology.Here, we propose a molecular taxonomy of MDS and describe its clinical relevance. Methods We assembled a cohort of 3,233 diagnostic MDS or related myeloid neoplasm samples from the International Working Group for the Prognosis of MDS. Gene mutations, copy-number alterations (CNAs) and copy-neutral loss of heterozygosity (cnLOH) events were derived from targeted sequencing of a 152-gene panel. Molecular features were used for unsupervised clustering analysis using Bayesian Dirichlet processes, and the results were manually curated into a rule-based hierarchical classification tree. Results We detected oncogenic mutations, CNAs, and cnLOH events in 91, 43, and 11% of patients, respectively. We validated and extended patterns of co-occurrence and mutual exclusivity between gene mutations and CNAs, and further described (i) specific occurrences in cis between gene mutations and cnLOH events, (ii) distinct patterns of co-mutation between the allelic state of a same locus (haploid LOH or cnLOH associated with -7/del(7q)), or of the same mutated gene ( TP53, TET2), and (iii) distinct patterns of co-mutation between hotspots of the same gene ( U2AF1). These patterns informed feature definition for clustering analysis. We characterized 18 distinct MDS molecular subgroups. The first 16 groups encompassed 86% (n=2,769) of patients and were based on the presence or absence of mutations in 21 genes ( ASXL1, BCOR, BCORL1, DDX41, DNMT3A, EZH2, FLT3, IDH1, IDH2, MLL, MYC, NPM1, SETBP1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2), and 6 cytogenetic events (inv(3), complex karyotype, der(1;7), -7, del(5q), -Y), as well as LOH at the TP53 and TET2 locus. The molecularly not-otherwise specified (mNOS) group(8%, n=254) corresponded to the presence of other cytogenetic abnormalities and/or mutations in 51 other recurrently mutated genes (>0.5%). The No-event category (6%, n=210) was characterized by the absence of any recurrent drivers in our assay. Groups ranged in size from 0.5% to 14% of patients and were associated with distinct clinical phenotypes ( Fig 1A) and outcomes ( Fig 1B). The median bone marrow blast percentage across groups ranged from 1.5 to 10%, the median overall survival (OS) from 0.9 to 8.2 years, and the 2-year rate of leukemic transformation from 0 to 40%. The representation of molecular groups within blast strata (<5%, 5-9%, 10-19% blast percentages) was highly heterogeneous: 7, 8, and 9 different groups accounted for more than 5% of patients within each blast stratum, respectively. The prognostic impact of blast counts on outcomes depended on the molecular subgroups. Among the 12 molecular groups with at least 10 patients with outcome data in all 3 blast strata, blast count was prognostically significant in 8 of those groups ( SF3B1, CCUS-like, del(5q), bi-TET2, mNOS, BCOR/L1, IDH-STAG2, TP53-complex) and was not significant in 4 groups ( EZH2-ASXL1, -7/SETBP1, DDX41, AML-like). The molecular groups validated established ( TP53-complex, del(5q), SF3B1) and well characterized entities ( DDX41, AML-like), added further evidence to support previously reported subsets ( bi-TET2, der(1;7), CCUS-like), and described novel genetic subsets (-7/ SETBP1, EZH2-ASXL1, IDH-STAG2, BCOR/L1, U2AF157, U2AF134, SRSF2, ZRSR2). Finally, the MDS molecular groups were used to investigate the genetic heterogeneity of secondary/therapy-related MDS (s/t-MDS) and of myelodysplastic/myeloproliferative neoplasms (MDS/MPN). There were 7 and 6 distinct groups that accounted for more that 5% of patients with s/t-MDS or MDS/MPN, respectively. Within each genetic subgroup, s/t-MDS and MDS/MPN had similar clinical and outcome profiles to primary MDS. Conclusions The genetic complexity of MDS can be organized into 18 molecular groups that reflect the underlying genetic basis of the disease. The prognostic influence of bone marrow blasts varied in the individual genetic subgroups, suggesting that the clinical impact of increased blasts depends on the genetic context. The molecular taxonomy derived in this study is clinically relevant and will inform future classification schemas.

UBA1 Mutations Identify a Rare but Distinct Subtype of Myelodysplastic Syndromes

Background Mutations in UBA1 are associated with VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, an adult-onset inflammatory disorder (Beck DB et al. NEJM 2020). Approximately 40% of VEXAS patients are also diagnosed with myelodysplastic syndromes (MDS). We previously profiled UBA1 p.M41 mutations in a select subset of the International Working Group for MDS (IWG-PM) cohort (Bernard E et al. NEJM Evid 2022) including 375 male patients lacking disease-defining mutations or established disease classification by WHO guidelines, and identified 28 patients (7%) with UBA1 p.M41T/V/L mutations (Cohort A, Sirenko M et al. Blood 2022). However, the prevalence and characteristics of UBA1 mutations in MDS have not been systematically evaluated in a representative MDS population. Methods We used targeted next-generation sequencing of the full UBA1 locus to profile 2027 diagnostic and treatment-naive MDS samples (Cohort B, 61% male) ascertained through the IWG-PM (Figure 1). Clinical associations of UBA1 mutations were evaluated and clinical history was reviewed for inflammatory conditions when available. Results In Cohort B, 35 UBA1 mutations in 34 (1.7%) patients were identified, of which 20 (0.97%) had likely pathogenic variants and 14 had variants of unknown significance (VUS). Likely pathogenic variants included 13 p.M41T/V/L mutation (variant allele fraction [VAF] range 0.013-0.94), and 7 non-p.M41 mutations where 6/7 had VAF>0.35: 2 with p.S56F (VAF 0.87 and 0.93), 2 p.A478S (VAF 0.72 and 0.80), 2 p.S621C (VAF 0.80 and 0.82) and 1 p.Y55H (VAF 0.025). Three VUS were in female patients. 204 cases were profiled by both assays and of those, all 8 ddPCR positive cases were identified by NGS with concordant VAF (R2=0.999 p<0.0001). Integration of Cohort A and B (n=2,198) yielded 40 patients (all male, median age 72; range 44-89 years) with pathogenic UBA1 variants. The WHO 2016 classification (available for n=38 of 40) was MDS-SLD/MLD (25), MDS-EB1 (4), MDS-U (3), CMML (2), MDS-RS-MLD (1), aCML (1), MDS/MPN-RS-T (1), MDS/MPN-U (1). Patients had a median of 1 myeloid gene mutation in addition to UBA1 (range: 0-4) with most frequent co-occurring events in TET2 (n=12), DNMT3A (n = 10), ASXL1 (n=3), SF3B1 (n=3) and loss of the Y chromosome (n=5). In 8 patients with pathogenic UBA1 > 2% VAF and co-occurring DNMT3A mutations, DNMT3A and UBA1 VAF were correlated (slope = 0.88, r = 0.87, p = 0.0005), suggesting that co-mutation may lead to clonal expansion. Conversely, TET2 co-mutations were either subclonal (n=4) or clonal (n=8) to UBA1. Among patients with pathogenic UBA1 variants, the majority had IPSS-M Very-Low/Low risk (73% 27/37). In contrast, patients with VUS were more likely to have Moderate or High risk (64% 7/11). Partial clinical history was available for 33 cases with pathogenic UBA1 variants. 50% (9/18) had inflammatory-rheumatic disease (IRD) including psoriatic arthritis, relapsing polychondritis, Sweet syndrome, bronchiolitis obliterans with organizing pneumonia, ear chondritis, and rosacea. 3 patients had more than one IRD. 8 were treated for IRD with steroids (n=6) or methotrexate (n=2). 4 patients had MGUS. Other manifestations included vacuoles (4/8), thromboembolic disease (5/16), non-infection fever (5/14), weight loss (4/14), ocular symptoms (5/15), arthralgia (3/16), chondritis (5/15), and other inflammation (6/14). IRD diagnosis usually preceded MDS (average time 0.28 years; range 16.5 years prior to 1.4 years after). Three patients transformed to acute myeloid leukemia (AML). One patient had a low VAF UBA1 p.M41V (0.0002 by ddPCR) and TET2, SF3B1, FLT3, and ASXL2 co-mutations at baseline. The second had UBA1 p.S56F (VAF 0.868) and a Chr 7q deletion. The third had VUS UBA1 p.R869L (VAF 0.222) and IRF1, NFE2 and RRAS co-mutations. Among the other UBA1-mutant patients that died or were censored after 1 year (n=37), none transformed to AML. Conclusion Within the large, representative, diagnostic and well-characterized IWG-PM MDS cohort, we find likely pathogenic UBA1 mutations in 1% of patients, with enrichment in male patients with few or no mutations in myeloid driver genes (7%). UBA1-mutant patients were predominantly IPSS-M low risk with a median of 1 additional mutation, usually in DNMT3A or TET2. UBA1 mutations may define a distinct subset of MDS and its recognition in future guidelines will improve the management of patients with MDS/VEXAS overlap.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Downregulation of Mitochondrial Complex II (MC II) in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of diseases of hematopoietic stem cells (HSCs) that occur in the aging population and following therapy for unrelated cancers. The standard of care for MDS involves hypomethylating agents (HMAs) that invariably result in resistance and disease progression. We previously determined the role of mitochondrial metabolism in MDS pathophysiology, whereby suppression of mitochondrial activity leads to the accumulation of oncometabolites that competitively inhibit α-ketoglutarate-dependent dioxygenases (2OGDDs). Consequently, hypermethylation of epigenomes and hypoxia-inducible factor 1A (HIF1A) activation ensues a pseudohypoxia state that drives MDS pathogenesis. Our recent informatics analyses of primary MDS samples identified a patient subset with high TGF-β signaling in which mitochondrial complex II (MC II) genes were downregulated. MC II, or succinate dehydrogenase (SDH) is the smallest complex of the oxidative phosphorylation system that oxidizes succinate to fumarate and thereby donates electrons to reduce FAD to FADH 2 and the ubiquinone pool. Its downregulation results in the accumulation of Krebs cycle intermediate metabolites that can act as antagonists for 2OGDDs. To understand whether SDH dysregulation alone can contribute to MDS pathobiology, we generated a doxycycline-inducible and reversible mouse model (SDH-KD) that expresses shRNAs against the SDH subunits (SDHA, B, and D) in HSCs. These mice showed a gradual buildup of intracellular succinate in c-Kit + bone marrow (BM) cells and the peripheral blood, and a corresponding decrease of fumarate levels after induction for 2 weeks, 4 weeks, and 2 months. We found that knocking down Sdh was sufficient to induce pancytopenia and trilineage dysplasia in SDH-KD mice, while intracellular iron granules that surround the nucleus of erythroblasts, similar to ring sideroblasts commonly observed in MDS patients were also detected in BM and spleen smears. Of note, removing doxycycline and restoring Sdh expression rescued macrocytic anemia among other MDS phenotypes. Furthermore, treating SDH-KD mice with HMAs restored mitochondrial respiration and reversed HIF1A activation through the upregulation of SDH subunits. Using another MDS model, Mll PTD/WT/ Vav1-Cre/Runx1 Flox/Flox, we found that HMA-treated HSCs differentiate towards CD11b +Gr1 + mature myeloid cells and that this requires Sdh expression. In conclusion, our results indicate that pseudohypoxia- and TGF-β signaling-associated changes in metabolic regulation caused by SDH downregulation underlie MDS pathogenesis. Elucidating the role of MC II in MDS pathobiology may provide alternative targets that can be combined with HMA or luspatercept therapy for MDS.

The Dual Inflammasome/Myddosome Inhibitor HT-6184 Restores Erythropoiesis in MDS/AML

Background: The NLRP3 inflammasome is one of the key biological drivers of ineffective hematopoiesis and inflammation in myelodysplastic syndrome (MDS). Inflammasome activation arises from both cell-extrinsic (e.g., Toll-like receptors) and cell-intrinsic signals (e.g., somatic mutations) that direct caspase-1 maturation leading to GATA-1 degradation, myeloid skewing, and pyroptotic cell death. HT-6184 is a potent allosteric inhibitor of the inflammasome scaffold protein NEK7 that blocks inflammasome assembly, promotes ASC speck dissolution, and inhibits myddosome activation of NFκβ. To investigate preclinical activity in MDS, we evaluated HT-6184 in MDS and acute myeloid leukemia (AML) models, including patient-derived sample cultures. Results: Expression of inflammasome pathway components was first assessed by gene expression profiling in a large cohort of MDS bone marrow CD34+-selected cells and healthy age-matched controls. Cellular expression of the inflammasome components, caspase-1, gasdermin D, IL-18 and Pycard was significantly upregulated in MDS specimens compared to healthy controls. Furthermore, cases with the highest expression of inflammasome effectors had significantly inferior overall survival (Log Rank P <0.05). The novel inflammasome inhibitor, HT-6184, was designed to prevent NEK7 from mediating the oligomerization of NLRP3 monomers following an activation signal, ultimately preventing inflammasome assembly. Thus, the structure of HT-6184 was tailored to maximize its interactions with NEK7, rigidifying the protein, and locking it in a conformation that prevents interaction with NLRP3. The allosteric activity of HT-6184 was examined using nano differential scanning fluorimetry and confirmed with limited proteolysis assays, which demonstrated that HT-6184 disrupts NEK7 conformations at single-digit nanomolar concentrations. This was confirmed in cell-based assays showing its ability to inhibit IL-1β release with a 50% inhibitory concentration (IC) of 46 nM in THP-1 cells. These data further correlate with HT-6184 blocking of ASC speck formation with an IC90 of 100 nM in THP-1 cells as well as speck dissolution. HT-6184 also affects the NF-κΒ pathway, by blocking myddosome function and resulting in a decreased expression of an NF-κΒ driven reporter gene, with an IC50 of 10.2 nM in transfected cells. HT-6184 exerts its pharmacological activity by inhibiting the priming and activation steps of the NLRP3 inflammasome. It is pharmacologically active in a human whole blood assay, strongly downregulating the production of pro-inflammatory cytokines. Anti-inflammatory efficacy was also observed in vivo in a murine inflammation model of inflammatory bowel disease. HT-6184 incubation with human monocytes abrogated production of IL-1β and IL-6, reverting human monocytes to a less inflamed state as demonstrated by single cell proteomic studies (isoplexis platform). It similarly decreased IL-1β production from monocytes derived from a TET2 -/+ mouse model that preferentially develops monocytic expansion. Finally, treatment initiation of primary MDS/AML bone marrow specimens (N=10) with HT-6184 in methylcellulose cultures markedly increased erythroid differentiation and reduced myelomonocytic colonies in the majority of samples. This was evidenced by the change in total number of colonies and by fluorescence activated cell sorting (FACS) assessment of differentiation markers (Glycophorin A, CD71, CD11b, CD14) after 14 days of incubation. Inflammasome-directed IL-1β secretion in primary MDS samples was also abrogated by HT-6184 treatment. Conclusions: These findings demonstrate that the novel dual inflammasome/myddosome inhibitor, HT-6184 effectively suppresses myeloid skewing, restores erythroid commitment in MDS/AML specimens, and provides a rationale for clinical development in myeloid malignancies.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMA) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5-azacytidine (5-AZA) could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in N=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene expression of the pro-apoptotic factor NOXA in ASXL1-mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

The CSN5/HSF/SPI1/PU.1 Axis Regulates Cell Proliferation in Hypocellular Myelodysplastic Syndrome Patients.

This study explored the relationship between the activation of the jak/stat3 signaling pathway and the CSN5 gene transcript and protein expression levels in the hematopoietic stem cells of patients with myelodysplastic syndromes (MDSs). This study also aimed to investigate the correlation between the expression level of CSN5 and the deubiquitination of HSF1, as well as the transcript level of the spi1/pu.1 genes to explore the pathogenesis of MDS. We isolated cells from normal individuals and MDS patients, and the mRNA and protein expression levels of spi1/pu.1 in cd34+ cells (hematopoietic stem cells) were measured by PCR and western blotting, respectively. A ChIP assay was used to detect the binding of HSF1 to the spi1/pu.1 promoter in cd34+ cells. The ubiquitination of HSF1 in cd34+ cells was detected by CO-IP. The binding of HSF1 and Fbxw7α was detected in in cd34+ cells by CO-IP. The binding of HSF1 and CSN5 was evaluated. A luciferase reporter assay was used to detect the effect of STAT3 on CSN5 promoter activation in cd34+ cells. Western blotting was used to detect the phosphorylation of STAT3 in cd34+ cells of MDS patients. The binding of STAT3 and C/EBP beta in cd34+ cells was detected by CO-IP. Inhibition of SPI1/PU.1 expression was observed in MDS samples with low proliferation ability. Further experiments proved that phosphorylation of STAT3 affected CSN5 function and mediated the ubiquitination of HSF, the upstream regulator of SPI1/PU.1 transcription, which led to the inhibition of SPI1/PU.1 expression. Restoration of CSN5 rescued the inhibition of HSF1 ubiquitination, causing SPI1/PU.1 transcription to resume and increasing SPI1/PU.1 expression, promoting the recovery of cell proliferation in hypocellular MDS. Our research revealed the regulatory role of the CSN5/HSF/SPI1/PU.1 axis in hypocellular MDS, providing a probable target for clinical intervention.

Progressive disruption of hematopoietic architecture from clonal hematopoiesis to MDS

Clonal hematopoiesis of indeterminate potential (CHIP) describes the age-related acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPC) leading to clonal blood cell expansion. Although CHIP mutations drive myeloid malignancies like myelodysplastic syndromes (MDS) it is unknown if clonal expansion is attributable to changes in cell type kinetics, or involves reorganization of the hematopoietic hierarchy. Using computational modeling we analyzed differentiation and proliferation kinetics of cultured hematopoietic stem cells (HSC) from 8 healthy individuals, 7 CHIP, and 10 MDS patients. While the standard hematopoietic hierarchy explained HSPC kinetics in healthy samples, 57% of CHIP and 70% of MDS samples were best described with alternative hierarchies. Deregulated kinetics were found at various HSPC compartments with high inter-individual heterogeneity in CHIP and MDS, while altered HSC rates were most relevant in MDS. Quantifying kinetic heterogeneity in detail, we show that reorganization of the HSPC compartment is already detectable in the premalignant CHIP state.

Analysis of core mutation and TET2/ASXL1 mutations DNA methylation profile in myelodysplastic syndrome

ABSTRACTObjectivesThe study aims to analyze genetic mutation and clinical characteristics and study their correlation with survival prognosis of patients with myelodysplastic syndromes (MDS). Moreover, the differential DNA methylation profiles between TET2 mutated (Mut)/ASXL1 wild-type (WT) and TET2-Mut/ASXL1-Mut MDS samples were investigated to explore the mechanism of MDS patients with TET2/ASXL1 mutations.MethodsThe clinical data of 195 patients diagnosed with MDS were selected and statistically analyzed. The DNA methylation sequencing data set was obtained from the GEO and bioinformatics analyzed.ResultsOf the 195 MDS patients, 42 (21.5%) carried TET2 mutations. 81% of TET2-Mut patients could detect comutated genes. The most commonly comutated gene in MDS patients with TET2-Mut was ASXL1, which had a tendency towards poorer prognosis (P = 0.08). GO analysis showed that highly methylated differentially methylated genes (DMGs) was mainly enriched in biological processes such as cell surface receptor signal pathway and cell secretion. Hypomethylated DMGs was mainly enriched in cell differentiation and cell development. KEGG analysis showed that hypermethylated DMGs was mainly enriched in Ras signal pathway and MAPK signal pathway. Hypomethylated DMGs was mainly enriched in extracellular matrix receptor interaction and focal adhesion. PPI network analysis identified 10 hub genes of hypermethylated and hypomethylated DMGs that may be associated with patients with TET2-Mut/ASXL1-Mut respectively.ConclusionsOur results illustrate the interrelationships between genetic mutations and clinical phenotypes and disease outcomes, with substantial potential for clinical application. Differentially methylated hub genes might represent potential biomarkers and provide novel insights and possible targets for MDS with double TET2/ASXL1 mutations.

Abstract 6168: Implementation and adoption of a web tool to support precision diagnostic and treatment decisions for patient with myelodysplastic syndromes

Abstract Despite a detailed understanding of the genes mutated in myelodysplastic syndromes (MDS), diagnostic and treatment decisions for patients with MDS rely primarily on clinical and cytogenetic variables as considered by the Revised International Prognostic Scoring System (IPSS-R). Here we describe the recently developed Molecular IPSS (IPSS-M), a clinico-genomic risk stratification system that considers clinical, cytogenetic and genetic parameters; the implementation of a web portal to facilitate its adoption, a strategy to handle missing variables, and the worldwide utilization of the web calculator as a clinical support tool. The IPSS-M was trained on 2,957 clinically annotated diagnostic MDS samples profiled for mutations in 156 driver genes. To maximize the clinical applicability of the IPSS-M and account for missing genetic data (i.e genes missing from a sequencing panel), we implemented a strategy to calculate a risk score under three scenarios: best, worst and average. Last, we developed an online calculator as a standalone single-page web application using VueJs, and D3Js for the interactive visualizations, deployed through a CI/CD pipeline on AWS, where collection of anonymous usage analytics allows to track adoption and usability of the new proposed model. The model incorporates clinical, morphological, genetic variables informed by cytogenetics and constructed from the presence of oncogenic mutations in 31 genes. It delivers a unique risk score for each individual patient, as well as an assignment to one of six IPSS-M risk strata. Compared to the IPSS-R the IPSS-M re-stratified 46% of MDS patients. The model was validated in an external dataset of 754 MDS patients. We released an open-access IPSS-M web calculator available at https://mds-risk-model.com. By specifying the patient clinical and molecular profiles, the tool returns the patient-specific IPSS-M risk score and category, and the probability estimates over time for three clinical endpoints, i.e. leukemia free survival (LFS), overall survival, and incidence of leukemic transformation. Since its launch in June 2022, the calculator has been used by >6000 users in >75 countries, reaching a daily average of 100 users per day. Risks have been calculated for >45,000 patient profiles. 99.28% of the sessions initiated reach an IPSS-M score, suggesting that the calculator is intuitive and easy to use. We trained and validated the IPSS-M on 3,711 patients, a patient tailored risk stratification tool for patients with MDS that considers clinical, morphological and genetic variables inclusive of cytogenetics and mutations in one of 31 genes. The development of a web based tool was instrumental to the global dissemination of the model, enabling non-expert users to leverage the power of molecular biomarkers in risk stratification for patients with MDS. Citation Format: Elsa Bernard, Juan E. Arango Ossa, Heinz Tuechler, Peter L. Greenberg, Robert P. Hasserjian, Yasuhito Nannya, Sean M. Devlin, Maria Creignou, Philippe Pinel, Lily Monier, Juan S. Medina-Martinez, Dylan Domenico, Martin Jädersten, Ulrich Germing, Guillermo Sanz, Arjan A. van de Loosdrecht, Olivier Kosmider, Matilde Y. Follo, Felicitas Thol, Lurdes Zamora, Ronald F. Pinheiro, Andrea Pellagatti, Detlef Haase, Pierre Fenaux, Monika Belickova, Michael R. Savona, Virginia M. Klimek, Fabio P. Santos, Jacqueline Boultwood, Ioannis Kotsianidis, Valeria Santini, Francesc Solé, Uwe Platzbecker, Michael Heuser, Peter Valent, Kazuma Ohyashiki, Carlo Finelli, Maria Teresa Voso, Lee-Yung Shih, Michaela Fontenay, Joop H. Jansen, José Cervera, Norbert Gattermann, Benjamin L. Ebert, Rafael Bejar, Luca Malcovati, Mario Cazzola, Seishi Ogawa, Eva Hellström-Lindberg, Elli Papaemmanuil. Implementation and adoption of a web tool to support precision diagnostic and treatment decisions for patient with myelodysplastic syndromes [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 6168.

Direct Comparison of NSG and NBSGW Mice for MDS Patient-Derived Xenograft Models

Introduction: Patient-derived xenograft (PDX) models of Myelodysplastic Syndromes (MDS) based on conditioned NSG mice are the most commonly used and well-established. Recently, our group has published an extensive pre-clinical study including n=49 MDS PDX by employing such a model (Schmitt et al., Leukemia 2022). However, achieving adequate engraftment rates and sufficient numbers of PDX can still be challenging, especially for low risk MDS patient samples. Moreover, human erythropoiesis is known to be severely impaired in NSG mice due to residual macrophage activity. Thus, there is a need for further optimization of MDS PDX models. The KIT-deficient mouse strains NBSGW and NSGW41 have already been shown to provide improved engraftment of cord blood and support of human erythropoiesis compared to NSG mice. (McIntosh et al., Stem Cell Reports 2015; Rahmig et al., Stem Cell Reports 2016). Therefore, in this study, we compared NSG and NBSGW mice of both sexes regarding human MDS engraftment, lineage differentiation, clonal bone marrow (BM) composition and survival. Methods: BM-derived CD34+ hematopoietic stem cells and autologous in-vitro-expanded mesenchymal stroma cells from n=5 MDS patients (n=3 MDS-MLD, n=2 MDS-EB-2) with varying IPSS-R scores (n=1 very low, n=1 low, n=1 intermediate, n=2 high) were intrafemorally co-transplanted into male and female busulfan-conditioned NSG and non-conditioned NBSGW mice. As controls, samples of n=2 age-matched, healthy (HY) donors were used. Human long-term engraftment (>0.01 %) was determined 12 weeks post transplantation. Endpoint evaluation was performed after 19-30 weeks. Peripheral blood (PB) and BM were analyzed for different human immunophenotypic cell populations including CD33, CD34, CD41, CD45, CD71 and CD235a. Further, human CD45+ cells from PDX BM were isolated by FACS for DNA extraction. Subsequently, extracted PDX and original patient samples were subjected to NGS panel sequencing of MDS-specific mutations. Results: Overall, our data showed that female and male NBSGW mice enabled higher MDS engraftment rates than NSG mice. Engraftment (% human CD45) of MDS and HY samples in NBSGW females was found to be considerably higher in BM and PB than in NSG females (BM: 17.2% (0.01-71.1%) in NSG mice versus 43.7% (1.1-87.7%) in NBSGW; PB: 1.5% (0.01-6.4%) in NSG mice versus 3.5% (0.03-18.4%) in NBSGW). Moreover, NBSGW mice showed improved support of human thrombopoiesis, as well as higher levels of erythropoiesis and CD34+ cells in both MDS and HY PDX compared to NSG mice. However, survival of NSG females was significantly longer than for NBSGW females (p=0.0024), as well as for NSG (p=0.0006) and NBSGW (p<0.0001) males. Interestingly, males of both strains showed lower engraftment rates and shorter overall survival in the experiment as compared to females. Observed early deaths of males were not associated with engraftment rates. Panel sequencing results of n=4 MDS patients and their corresponding PDX showed no differences between NSG and NBSGW mice in mutational profiles and variant allele frequencies of transplanted MDS samples. Both strains were able to faithfully recapitulate the patient's clonal BM composition. Conclusions: Female NBSGW mice are superior engrafters for primary MDS samples in comparison to female NSG mice and provide the opportunity to investigate human thrombopoiesis and erythropoiesis in-vivo. While NSG PDX were already proven to faithfully recapitulate the clonal BM composition of transplanted patient samples, we found that the same is true for NBSGW mice. However, female NSG mice were more robust in terms of survival. This has to be taken into account when designing long-term PDX studies. Finally, in our study, male mice of both strains were shown to be inferior acceptors for PDX as compared to females.