Del 5q Myelodysplastic Syndrome Research Articles

Abstract 248: HSPA9/mortalin regulates erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells

Abstract Mortalin, encoded by the HSPA9 gene, is a highly conserved heat-shock chaperone belonging to the HSP70 family. It is predominantly presented in the mitochondria, and is critical in regulating a variety of cell physiological functions such as response to cell stress, control of cell proliferation, and inhibition/prevention of apoptosis. Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell malignancies characterized by ineffective hematopoiesis, increased apoptosis of bone marrow cells, and anemia. Up to 25% of MDS patients harbor an interstitial deletion on the long arm of chromosome 5, also known as del(5q), creating haploinsufficiency for multiple genes including HSPA9. Our prior study showed that knockdown of HSPA9 induces TP53-dependent apoptosis in human CD34+ hematopoietic progenitor cells, consistent with cytopenia observed in MDS patients. Since anemia is another featured symptom of MDS, we hypothesize that HSPA9 plays a role in regulating erythroid maturation. To test our hypothesis, we inhibited the expression of HSPA9 using various methods and measured the erythroid maturation in human CD34+ cells. We used siRNA targeting HSPA9 and found that HSPA9 siRNA significantly inhibited the cell growth, increased cell apoptosis, inhibited erythroid maturation (using CD71 as a surrogate marker), and increased p53 expression (p<0.01) compared to control siRNA in human CD34+ cells. Pharmacologic inhibition of HSPA9 by the chemical MKT-077, an inhibitor of HSP70 protein family members including mortalin, also increased p53 expression and inhibited erythroid maturation in human CD34+ cells. In addition, knockdown of HSPA9 by shRNA showed significant inhibition of erythroid maturation in human CD34+ cells compared to control shRNA. In order to test whether the regulation of erythroid maturation by HSPA9 is TP53-dependent or not, we constructed shRNAs targeting TP53 genes and simultaneously transduced lentivirus containing shRNAs targeting HSPA9 and TP53 respectively in human CD34+ cells using double antibiotics selection (puromycin for shRNA targeting HSPA9 and hygromycin for shRNA targeting TP53). We found that TP53 knockdown partially rescued the erythroid maturation defect caused by HSPA9 inhibition, suggesting that erythroid maturation inhibition by HSPA9 knockdown is partly mediated through a TP53 mechanism. Collectively, our results suggest that the increased apoptosis and reduced erythroid maturation observed in del(5a)-associated MDS is TP53-dependent. HSPA9/mortalin may be a potential target to treat anemia in del(5q) MDS patients, although simultaneous loss of multiple genes on del(5q) likely contributes to the complex phenotypes observed in MDS. Thus, our study not only uncovers some underlying mechanisms of del(5q) MDS, but also provides potential therapeutic indications through gene targeting in clinical MDS treatment. Citation Format: Tuoen Liu, Christopher Butler, Morgan Dunmire, Gabor Szalai, Anissa Johnson, Jaebok Choi, Matthew Walter. HSPA9/mortalin regulates erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells [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 248.

Collaborative effect of Csnk1a1 haploinsufficiency and mutant p53 in Myc induction can promote leukemic transformation

AbstractIt is still not fully understood how genetic haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) contributes to malignant transformation of hematopoietic stem cells. We asked how compound haploinsufficiency for Csnk1a1 and Egr1 in the common deleted region on chromosome 5 affects hematopoietic stem cells. Additionally, Trp53 was disrupted as the most frequently comutated gene in del(5q) MDS using CRISPR/Cas9 editing in hematopoietic progenitors of wild-type (WT), Csnk1a1–/+, Egr1–/+, Csnk1a1/Egr1–/+ mice. A transplantable acute leukemia only developed in the Csnk1a1–/+Trp53–edited recipient. Isolated blasts were indefinitely cultured ex vivo and gave rise to leukemia after transplantation, providing a tool to study disease mechanisms or perform drug screenings. In a small-scale drug screening, the collaborative effect of Csnk1a1 haploinsufficiency and Trp53 sensitized blasts to the CSNK1 inhibitor A51 relative to WT or Csnk1a1 haploinsufficient cells. In vivo, A51 treatment significantly reduced blast counts in Csnk1a1 haploinsufficient/Trp53 acute leukemias and restored hematopoiesis in the bone marrow. Transcriptomics on blasts and their normal counterparts showed that the derived leukemia was driven by MAPK and Myc upregulation downstream of Csnk1a1 haploinsufficiency cooperating with a downregulated p53 axis. A collaborative effect of Csnk1a1 haploinsufficiency and p53 loss on MAPK and Myc upregulation was confirmed on the protein level. Downregulation of Myc protein expression correlated with efficient elimination of blasts in A51 treatment. The “Myc signature” closely resembled the transcriptional profile of patients with del(5q) MDS with TP53 mutation.

Foxm1 haploinsufficiency drives clonal hematopoiesis and promotes a stress-related transition to hematologic malignancy in mice.

Clonal hematopoiesis plays a critical role in the initiation and development of hematologic malignancies. In patients with del(5q) myelodysplastic syndrome (MDS), the transcription factor FOXM1 is frequently downregulated in CD34+ cells. In this study, we demonstrated that Foxm1 haploinsufficiency disturbed normal hematopoiesis and conferred a competitive repopulation advantage for a short period. However, it impaired the long-term self-renewal capacity of hematopoietic stem cells, recapitulating the phenotypes of abnormal hematopoietic stem cells observed in patients with MDS. Moreover, heterozygous inactivation of Foxm1 led to an increase in DNA damage in hematopoietic stem/progenitor cells (HSPCs). Foxm1 haploinsufficiency induced hematopoietic dysplasia in a mouse model with LPS-induced chronic inflammation and accelerated AML-ETO9a-mediated leukemogenesis. We have also identified Parp1, an important enzyme that responds to various types of DNA damage, as a target of Foxm1. Foxm1 haploinsufficiency decreased the ability of HSPCs to efficiently repair DNA damage by downregulating Parp1 expression. Our findings suggest that the downregulation of the Foxm1-Parp1 molecular axis may promote clonal hematopoiesis and reduce genome stability, contributing to del(5q) MDS pathogenesis.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

MDS-454 and Mutations Might Play a Role in Poor Prognosis and AML Progression in Patients With Isolated del(5q) MDS

Myelodysplastic syndromes (MDS) with isolated del(5q) represent a subtype defined by WHO with good prognosis. However, several studies have shown that the presence of somatic mutation, such as TP53, might have an impact on prognosis. In addition, recent publications suggest a role for SF3B1 in the poor outcome of some del(5q) MDS patients. Our aim was to validate the poor prognosis of SF3B1 mutations, present in patients with del(5q) MDS and to identify mutational profiles that could promote AML progression of patients del(5q) MDS. We performed a retrospective study including MDS patients with both isolated del(5q) and del(5q) with another alteration different from chromosome 7. We included 50 patients and a control group of 123 low-risk MDS patients with normal karyotype. Next-generation sequencing (NGS) data from bone marrow samples were analyzed using an in-house custom panel (>100 myeloid genes). Median age at diagnosis was similar in del(5q) MDS and control group (72.9 and 71.8 years, respectively, p>0.05). The number of mutations per patient was significantly higher in the control group than in del(5q) (a mean of 3.16 against 1.7 for del(5q), p<0.0001). Despite a higher prevalence of somatic mutations in the control group, no differences were found in median overall survival (OS) (64.5 and 60.1 months for del(5q) MDS and control group respectively, p=0.413). However, when analyzing del(5q) patients individually, OS was significantly lower in those patients with either TP53 or SF3B1 mutation (64.5 vs. 28 months median) (p=0.031). Regarding progression to AML, patients with TP53 and/or DNMT3A mutations (n=14) had a shorter time to progression compared to patients without mutations in these genes, with a median of 13.6 months vs. 26.97 months (p=0.004). Median follow-up for these patients was 14.93 (0-170.30) months. MDS patients with del(5q) have a good prognosis and a higher time to progression to AML compared to other MDS subgroups. However, the mutational profile may modify this evolution. In our study, mutations in SF3B1 and DNMT3A may be involved in OS and transformation to AML (respectively), but a larger cohort of patients is needed to confirm that.

MDS-450 Impact of Number of Cells Harboring Deletion 5q Abnormality by Conventional Cytogenetics in Isolated Deletion 5q Myelodysplastic Syndromes

Isolated del 5q myelodysplastic syndrome (MDS) is defined as presence of del 5q +/- one additional chromosomal abnormality without excess myeloblasts. Lenalidomide (len) is current standard of care for those patients. The impact of percentage of cells (number of metaphases) harboring del 5q by karyotype is not known. We examined our cohort of isolated del 5q MDS patients at Moffitt Cancer Center. We assessed baseline characteristics, response to len, and outcomes based on % of cells/metaphases harboring del 5q using G-banding "conventional cytogenetics clone size." We divided patients into 2 groups based on number of cells harboring del 5q, those with <50% i.e less than 10 out 20 metaphases and 50-100% (10 or more out of 20 metaphases). We identified 142 isolated del 5q MDS patients. Baseline characteristics of the whole cohort was similar to what has been previously described in the literature. Patients with >50% metaphases harboring del 5q had more severe anemia (Hgb < 8.0 g/dl) (30% versus 13%) (p=0.05). Among patients who were treated with len (n=100), 50% achieved a response. There was a trend for higher response rate among patients with >50% del 5q metaphases with (42/80) 53% response rate compared to (8/20) 40% among those with <50%. (p=0.32). There was no difference in presence of TP53 somatic mutation based on % of metaphases harboring del 5q. There was a non-statistically significant trend for better overall survival among patients with >50% cells with detected del 5q compared to those with <50%, median OS 57 mo versus 35 mo, p=0.90. The number of cells harboring del 5q among isolated del 5q MDS patients using regular karyotyping "clone size by conventional cytogenetics" correlated with severity of anemia, trend for better response to len and trend for better overall survival. Those findings should be further confirmed in larger cohort of patients.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Genetic barcoding systematically compares genes in del(5q) MDS and reveals a central role for CSNK1A1 in clonal expansion

AbstractHow genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1−/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1−/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

Therapeutic Outcomes and Prognostic Impact of Gene Mutations Including TP53 and SF3B1 in Patients with Del(5q) Myelodysplastic Syndromes (MDS)

BackgroundGenetic alterations are increasingly being recognized to play an important role in both diagnosis and prognosis of MDS. In general, MDS patients with SF3B1 mutations (MT) are known to have favorable outcomes whereas those with TP53 mutations have dismal survivals. However, it is unclear if the impact of these mutations applies to all subtypes of MDS including del(5q) which is known for its response to lenalidomide and better prognosis. Materials and MethodsWe retrospectively reviewed 132 del(5q) MDS patients who were treated at the Moffitt Cancer Center (2001-2019). ResultsAmong patients who received lenalidomide (n = 98), 50%, 42.9%, and 7.1% achieved hematologic improvement or better, no response, and disease progression/death with a median overall survival (mOS) of 93.2, 72.4, and 25.6 months, respectively (P < .0001). The mOS was 73.3 months but only 25.6 months after patients stopped lenalidomide. TP53 was the most common mutation accounting for 23.8% of the patients. Of the 63 patients with molecular data available, 23.8% harbored TP53 MT and 10% with SF3B1 MT. TP53 status did not impact OS (MT 86.4 vs. wild-type (WT) 73.3 months; P = .72) but those with SF3B1 mutations had a significantly shorter mOS compared to WT (23.9 vs. 83.5 months; P = .001). Multivariate analysis confirmed lenalidomide response and SF3B1 mutations are independently associated with outcomes. ConclusionOur findings indicate many del(5q) MDS patients will benefit from lenalidomide but survival after its failure is limited. Mutations known to have prognostic impact in MDS at large may not have the same implications in the del(5q) subset.

Lenalidomide for anemia correction in lower-risk del(5q) myelodysplastic syndrome patients of Asian ethnicity.

BackgroundTo estimate real-world outcomes in East Asian populations, we conducted a nationwide retrospective analysis of the efficacy and safety of lenalidomide for del(5q) myelodysplastic syndrome (MDS) patients with transfusion-dependent anemia in Korea.MethodsPatients aged ≥19 years who had received lenalidomide for the treatment of lower-risk, red blood cell (RBC) transfusion-dependent del(5q) MDS were selected. A filled case report form (CRF) with information from electronic medical records was requested from members of the acute myeloid leukemia (AML)/MDS Working Party of the Korean Society of Hematology. All the CRFs were gathered and analyzed.ResultsA total of 31 patients were included in this study. Of 28 evaluable patients, 19 (67.9%) achieved RBC transfusion independence (RBC-TI). Female sex and the development of thrombocytopenia during treatment were associated with achieving RBC-TI. The most common non-hematologic toxicities were pruritus, fatigue, and rashes. All non-hematologic toxicities of grades ≥3 were limited to rash (12.9%) and pruritus (6.5%). Dose reduction was required in 15 of the 19 responders (78.9%). The most common final stable dosing schedule for the responders was 5 mg once every other day (31.6%).ConclusionLenalidomide efficacy and tolerability were similar in the Asian del(5q) MDS patients and western patients. Dose reduction during treatment was common, but it was not associated with inferior outcomes.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

Tifab Deficiency Protects Against Stress-Induced Bone Marrow Failure at the Expense of Pre-Leukemic Selection

Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. TRAF-interacting protein with forkhead-associated domain B (TIFAB) resides within the proximal commonly-deleted region on band 5q31.1, and belongs to a family of forkhead-associated domain proteins. TIFAB is deleted in nearly all reported cases of del(5q) MDS and AML. As expected, TIFAB expression is significantly lower in CD34+ and BM mononuclear cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q. Recently we have shown that hematopoietic-specific deletion of Tifab in mice results in progressive BM and blood defects, including aberrant HSPC proportions, altered myeloid differentiation, and progressive cytopenia (Varney and Niederkorn et al., JEM 2015). Gene expression analysis of Tifab KO lineage-Sca1+cKit+ (LSK) cells identified dysregulation of immune-related signatures, hypersensitivity to Toll-like receptor stimulation, and enrichment of a p53 regulatory gene-set.Importantly, p53 is implicated in the pathogenesis of del(5q) MDS: 1) BM cells from murine models and BM from del(5q) patients exhibit increased p53 activity, which is thought to contribute to ineffective hematopoiesis and anemia; and 2) del(5q) MDS patients often acquire concurrent TP53 mutations that result in rapid transformation to AML and poor treatment response. To examine the effects of TIFAB on p53 function, we examined p53 target genes in TIFAB-overexpressing and -deficient cells. Gene expression profiling and qRT-PCR analysis of Tifab KOHSPCs revealed significant upregulation of p53 regulatory genes. In contrast, overexpression of TIFAB in a p53-competent cell line reduced the expression of p53 target gene, p21 . We also observed increased p53 protein in Tifab KO total BM, lineage- BM, and spleen. At steady-state and after in vitro treatment with 2 Gy irradiation, Tifab KO lineage- BM cells exhibit increased p53 protein compared to WT cells.To test whether this increased p53 stability renders Tifab -deficient HSPCs more sensitive to stress, we treated Tifab KO HSPCs both in vivo and ex vivo using irradiation. When treated with low dose irradiation ex vivo, Tifab KO HSPCs initially exhibit significantly reduced colony forming ability; however, upon secondary re-plating, Tifab KO HSPCs strikingly form more colonies than WT HSPC, resembling a clonal selection event. To model this effect in vivo, we transplanted WT or Tifab KO BM into lethally-irradiated recipient mice. After 4 weeks of hematopoietic reconstitution, recipient mice were treated with 6.5 Gy sublethal irradiation. We find that Tifab KO recipient mice are less likely to succumb to IR-induced bone marrow failure as compared to mice transplanted with WT BM cells. Instead, after 3+ months, the remaining Tifab KO recipient mice have an increased tendency to develop hematologic malignancies, evident by myeloid blasts. Collectively, these data suggest that TIFAB-deficiency protects against cellular stress by eliminating “less fit” HSPCs, at the expense of selecting for pre-leukemic populations, paralleling observations in patients with transformed MDS.To investigate the mechanism of this phenomenon, we performed a tandem-affinity tag purification and mass-spectrometry analysis of TIFAB complexes in a del(5q) AML cell line (HL60), and identified unique TIFAB-interacting proteins. The top interacting candidate was an ubiquitin-specific peptidase (USP), USP15. USPs play a major role in ubiquitin-dependent processes including DNA damage response signaling, protein degradation, and kinase activation. Specifically, USP15 has been shown to promote p53 degradation via deubiquitination and stabilization of its major negative regulator, MDM2. Through biochemical assays and a series of deletion mutants, we confirmed that TIFAB interacts with USP15 and enhances the deubiquitination of biological USP15 substrates, including MDM2. Thus, our data implicate TIFAB as an important negative regulator of p53 and modulator of USP15 DUB activity.Altogether, our findings identify a novel role for TIFAB as a modifier of p53 function, potentially via a USP15-MDM2 axis. These findings have important implications in the potential role of TIFAB and p53 signaling in the pathogenesis of del(5q) MDS and transformation to AML. DisclosuresNo relevant conflicts of interest to declare.

Innate Immune Signal Pathway Activation Induced By Combined Loss of Del(5q) MDS Genes, Tifab and Mir-146a, Accelerates Hematopoietic Stem Cell Exhaustion Driven By Chronic Exposure to a TLR Ligands

Deletion of the long arm of chromosome 5 (del(5q)) is the most frequently observed chromosomal abnormality in patients with myelodysplastic syndrome (MDS). We previously reported that deletion of TRAF-interacting protein with forkhead-associated domain B (TIFAB) and miR-146a, two haploinsufficient genes in del(5q) MDS, activates the Toll-like receptor (TLR) signaling cascade in hematopoietic stem/progenitor cells (HSPC) by increasing TRAF6 protein stability and mRNA translation, respectively (Varney et al., JEM 2015 and Varney et al., Leukemia 2017). To investigate the epistasis of TIFAB and miR-146a, we generated a mouse model in which Tifab and miR-146a were simultaneously deleted (Tifab-/-; miR-146a-/-, herein dKO). Consistent with increased TRAF6 expression, dKO mice exhibit phosphorylation of IRAK1 and IRAK4 kinases, indicating activation of the TLR/innate immune pathway in bone marrow (BM) HSPC of dKO mice. To determine whether combined deletion of Tifab and miR-146a exaggerates the hematopoietic-specific defects observed in either Tifab- or miR-146a-deficient mice in vivo, BM cells from WT, Tifab-/-, miR-146a-/-, and dKO mice were transplanted into lethally-irradiated syngeneic recipient mice. As previously reported, a minor subset of Tifab-/- and ~ 50% of miR-146a-/- mice succumbed to a bone marrow failure (BMF)-like disease. For dKO mice, 80% of mice developed a more rapid and penetrant BMF-like disease as compared to loss of Tifab or miR-146a alone. Moribund dKO mice presented with leukopenia, severe anemia, and thrombocytopenia. Since TLR activation is a common feature of MDS due to overexpression and secretion of damage-associated molecular patterns (DAMPs) within the BM microenvironment (Monlish et al., Front Immunol 2016), we explored the biological effect of systemic TLR activation on hematopoietic stem cell (HSC) function of dKO mice following exposure to chronic low dose lipopolysaccharide (LPS), a TLR4 agonist. After 4 weeks, the proportion of myeloid cells in peripheral blood (PB) of LPS-treated dKO mice was significantly increased as compared to WT mice exposed to LPS. BM cellularity of dKO mice was dramatically decreased by LPS exposure while that of WT mice was only slightly affected by LPS. The proportion of CD48-CD150+Lineage-Sca1+c-Kit+ (LSK) of dKO mice was significantly reduced by exposure to LPS as compared to WT mice, which correlated with loss of HSC quiescence. We next performed competitive repopulation assays using WT and dKO BM cells exposed to LPS. As expected, the chimerism of donor-derived PB cells from LPS-treated WT mice was significantly lower than that from WT control mice. Intriguingly, LPS-treated dKO mice showed significantly reduced chimerism of donor-derived PB and CD48-CD150+ LSK BM cells as compared to vehicle-treated dKO mice or WT mice exposed to LPS. Importantly, the impaired hematopoietic reconstitution was more pronounced following serial BM transplantation. These results indicate that deletion of Tifab and miR-146a diminishes the long-term repopulating potential of HSC driven by in vivo TLR stimulation. To uncover the molecular consequences that explain the functional HSC defect of dKO mice exposed to TLR activation, RNA-sequencing was performed on LSK from dKO and WT mice treated with LPS. Following TLR activation, we observed differential expression of distinct genes in dKO LSK as compared to WT LSK. Pathway network analyses revealed changes in chromatin organization, epigenetic regulations, and cellular senescence in WT LSK exposed to LPS as compared to control WT cells. In contrast, dKO LSK exposed to LPS exhibited enrichment in pathways associated with axon guidance, carbohydrate metabolism, and megakaryocyte development and platelet production as compared to control dKO cells. These results suggest the differential impact of TLR stimulation on hematopoiesis in Tifab;miR-146a-deficient mice. Collectively, our findings indicate that innate immune signal pathway activation induced by combined loss of Tifab and miR-146a accelerates HSC exhaustion driven by chronic exposure to TLR ligands via the activation of distinct signal pathways. These findings have important implications in the role of TLR stimulation in the cellular consequence of MDS HSC. DisclosuresNo relevant conflicts of interest to declare.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

Blockade of IL-22 signaling reverses erythroid dysfunction in stress-induced anemias.

Patients with myelodysplastic syndromes (MDS) display severe anemia but the mechanisms underlying this phenotype are incompletely understood. Right open-reading-frame kinase 2 (RIOK2) encodes a protein kinase located at 5q15, a region frequently lost in MDS del(5q) patients. Here, we show that hematopoietic cell-specific haploinsufficient deletion of Riok2 (Riok2f/+Vav1cre) led to reduced erythroid precursor frequency leading to anemia. Proteomic analysis of Riok2f/+Vav1cre erythroid precursors suggested immune system activation and transcriptomic analysis revealed an increase in p53-dependent interleukin-22 (IL-22) in Riok2f/+Vav1cre CD4+ T cells (TH22). Further, we discovered that the IL-22 receptor, IL-22RA1, was unexpectedly present on erythroid precursors. Blockade of IL-22 signaling alleviated anemia not only in Riok2f/+Vav1cre mice but also in wild-type mice. Serum concentrations of IL-22 were increased in the subset of del(5q) MDS patients as well as patients with anemia secondary to chronic kidney disease (CKD). This work reveals a possible therapeutic opportunity for reversing many stress-induced anemias by targeting IL-22 signaling.

SF3B1 Mutations and Not TP53 Are Associated with Poor Outcomes in Patients with Del(5q) Myelodysplastic Syndromes (MDS)

Background: Genetic alterations are increasingly being recognized to play an important role in both diagnosis and prognosis of MDS. In general, SF3B1 mutated MDS is known to have favorable outcomes whereas those with mutated TP53 have dismal survivals. However, it is unclear if the impact of these mutations applies to all subtypes of MDS including del(5q), a unique entity that is known for its sensitivity to lenalidomide and better prognosis. A prior study suggests TP53 mutations are associated with worse outcomes in del(5q) MDS (Jadersten et al. 2011), but a more recent study did not find this association and instead they showed SF3B1 mutants (MT) conferred shorter survival compared to wild-type (WT) (Meggendorfer et al. 2017). Herein, we describe our experience of del(5q) MDS patients and assess the prognostic impact of somatic mutations in this population. Methods: We retrospectively reviewed our database of 132 patients with del(5q) MDS who were treated at the Moffitt Cancer Center from 2001 to 2019. Sixty-three patients had molecular data available for analysis. Fisher's exact test of independence was used to determine significance for categorical variables. Univariate and multivariate analyses were conducted using log-rank and Cox regression, respectively. Kaplan-Meier analysis with log-rank test was used to estimate survival outcomes. Results: A total of 132 patients (47M/85F) with a median age of 70 at diagnosis were included in this analysis (Table 1). The majority had isolated del(5q) (86.3%) and the remaining with del(5q) plus other aberration(s) but not meeting criteria as complex cytogenetics. Among patients who received lenalidomide (n=98), 50%, 42.9%, and 7.1% achieved hematologic improvement (HI) or better, no response (NR), and disease progression (DP)/death with a median overall survival (OS) of 93.2, 72.4, and 25.6 months from the time of diagnosis, respectively (p&amp;lt;0.0001) (Figure 1). The median OS of the entire cohort was 73.3 months (median follow-up of 131.3 months), and OS from the time patients stopped lenalidomide was only 26 months. Of the 63 patients with molecular data available, 23.8% were found to have TP53 mutations with a median variant allele frequency (VAF) of 20.4% and 10% SF3B1 MT (median VAF 38.9%). Other common mutations include TET2 (19%) and DNMT3A (17%) as shown in Figure 2. Interestingly, TP53 status did not impact OS (MT 86.4 vs. WT 73.3 months; p=0.72), but those with SF3B1 mutations had a significantly shorter median OS compared to WT (23.9 vs. 83.5 months; p=0.001) (Figure 3). When test for effects of multiple genes had on survival, no combinations were found to be predictive. Univariate analysis showed lenalidomide response significantly impacted OS (HI compared to NR: HR 0.19, 95% CI: 0.05-0.68, p=0.005). Multivariate regression confirmed lenalidomide response and SF3B1 status are independently associated with outcomes. Conclusions: SF3B1 mutations, typically correlate with favorable risk in MDS, are associated with significantly worse prognosis compared to WT in patients with del(5q) MDS. On the contrary, there was no significant difference in OS in patients with TP53 MT vs. TP53 WT. A substantial portion of patients benefited from lenalidomide; however, after lenalidomide failure survivals were limited. Despite the known overall associations between MDS and mutations such as SF3B1 and TP53, our data suggests the del(5q) subset behaves differently which adds to the heterogeneity of the disease. Further studies are needed to clarify the impact of treatments and somatic mutations on outcomes. Disclosures Sallman: Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy; Celgene, Jazz Pharma: Research Funding. Padron:Novartis: Honoraria; Incyte: Research Funding; Kura: Research Funding; BMS: Research Funding. Lancet:Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Komrokji:BMS: Honoraria, Speakers Bureau; Agios: Speakers Bureau; Abbvie: Honoraria; Incyte: Honoraria; Acceleron: Honoraria; Novartis: Honoraria; Jazz: Honoraria, Speakers Bureau; Geron: Honoraria.

IL-6 Deficiency Reverses Leukemic Transformation in an MDS Mouse Model

Myelodysplastic syndromes (MDS) are a group of age-related myeloid malignancies that are characterized by ineffective hematopoiesis and increased incidence of developing acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, which is partially due to the scarcity of leukemia transformation mouse models. Recently, we established a mDia1/miR146a double knockout (DKO) mouse model mimicking human del(5q) MDS. DKO mice present with pancytopenia with aging due to myeloid suppressive cell (MDSC) expansion and over-secretion of pro-inflammatory cytokines including TNF-a and interlukine-6 (IL-6). In the current study, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. The bone marrow of these mice was largely replaced by c-Kit+ blasts in a background of fibrosis. Flow cytometry analysis and in vitro colony formation assay demonstrated that hematopoietic stem progenitor cells (HSPCs) in DKO bone marrow were dramatically declined. The leukemic DKO mice had elevated white blood cell counts and circulating blasts, which contributes to the myeloid cell infiltration in non-hematopoietic organs including liver and lung. Moreover, the splenocytes from DKO old mice efficiently reconstitute the hematopoiesis, but led to a 100% disease occurrence with rapid lethality in gramma irradiated recipient mice, suggesting the leukemic stem cells enriched in DKO spleen were transplantable. Given the significant roles of the inflammatory cytokines in the pathogenesis of the DKO mice, we crossed DKO mice with IL-6 knockout mice and generated mDia1/miR-146a/IL-6 triple knockout (TKO) mice. Strikingly, the TKO mice showed dramatic rescue of the leukemic transformation of the DKO mice in that all the aforementioned leukemic phenotypes were abolished. In addition, IL-6 deficiency normalized the cell comparts and prevented leukemic transplantation ability in TKO spleen. Single cell RNA sequencing analyses indicated that DKO leukemic mice had increased monocytic blast population with upregulation of Fn1, Csf1r, and Lgals1, that was completely diminished with IL-6 knockout. Through a multiplex ELISA, we found IL-6 deficiency attenuated the levels of multiple inflammatory cytokines in TKO serum. In summary, we report a mouse model with MDS leukemic transformation during aging, which could be reverted with the depletion of IL-6. Our data indicate that IL-6 could be a potential target in high risk MDS. Disclosures No relevant conflicts of interest to declare.

MDS-372: TP53 Mutated del(5q) Myelodysplastic Syndromes (MDS) Patients Have Survivals Comparable to Those with TP53 Wild-Type

Background: TP53 mutations confer dismal prognosis in many hematologic malignancies, including MDS in general. It is frequently found in higher-risk/therapy-related MDS. However, 15-20% can occur in patients with del(5q), a subgroup that is known to have a more favorable risk. A prior study suggests TP53 mutations are associated with worse outcomes in del(5q) MDS. Objective: To assess the prognostic impact of TP53 mutations in del(5q) MDS patients and to report response to lenalidomide. Methods: This is a retrospective study of del(5q) MDS patients who were treated at the Moffitt Cancer Center from 2001 to 2019. Out of 132 patients identified, 63 had molecular data available and represent the cohort of present study. Univariate (log-rank), multivariate (Cox regression), and Kaplan-Meier (log-rank) analyses were performed. Results: Of the 63 patients (26M/37F), 23.8% were found to have TP53 mutation. Majority had isolated del(5q) (85%) with the remaining del(5q) plus other aberration(s) but not meeting criteria as complex. A total of 43 patients (68.3%) received lenalidomide (73.3% TP53 MT and 66.7% TP53 WT, p=NS) with 37.2%, 55.8%, and 7% achieving HI, NR, and DP/death, respectively. AML transformation occurred in 14.3% (20% TP53 MT and 12.5% TP53 WT, p=NS). Other common mutations include TET2 (19%), DNMT3A (17%), and SF3B1 (10%). The median OS was 76.6 months (median follow-up: 71.3 months). There was no significant difference in OS regardless of TP53 mutational status (p=0.72). Among patients who received lenalidomide, mOS for HI, NR, and DP/death were 142.2, 66, and 25.6 months, respectively (p ≤ 0.001). Univariate analysis showed lenalidomide response significantly impacted OS (HI:HR 0.02, 95% CI: 0.00-0.11, p ≤ 0.001; NR:HR 0.08, 95% CI: 0.02-0.43, p=0.003). Multivariate regression using covariates including TP53 status confirmed lenalidomide response to be independently associated with outcomes. Conclusions: There was no significant difference in OS in patients with TP53 MT vs. TP53 WT. A substantial portion of del(5q) MDS patients benefited from lenalidomide. Despite the known overall adverse association between TP53 and MDS, our data suggests the del(5q) subset behaves differently, which adds to the heterogeneity of the disease. Further studies are needed to clarify the impact of treatments and somatic mutations on outcomes.

PRC2 insufficiency causes p53-dependent dyserythropoiesis in myelodysplastic syndrome

EZH1 and EZH2 are enzymatic components of polycomb repressive complex (PRC) 2, which catalyzes histone H3K27 tri-methylation (H3K27me3) to repress the transcription of PRC2 target genes. We previously reported that the hematopoietic cell-specific Ezh2 deletion (Ezh2Δ/Δ) induced a myelodysplastic syndrome (MDS)-like disease in mice. We herein demonstrated that severe PRC2 insufficiency induced by the deletion of one allele Ezh1 in Ezh2-deficient mice (Ezh1+/-Ezh2Δ/Δ) caused advanced dyserythropoiesis accompanied by a differentiation block and enhanced apoptosis in erythroblasts. p53, which is activated by impaired ribosome biogenesis in del(5q) MDS, was specifically activated in erythroblasts, but not in hematopoietic stem or progenitor cells in Ezh1+/-Ezh2Δ/Δ mice. Cdkn2a, a major PRC2 target encoding p19Arf, which activates p53 by inhibiting MDM2 E3 ubiquitin ligase, was de-repressed in Ezh1+/-Ezh2Δ/Δ erythroblasts. The deletion of Cdkn2a as well as p53 rescued dyserythropoiesis in Ezh1+/-Ezh2Δ/Δ mice, indicating that PRC2 insufficiency caused p53-dependent dyserythropoiesis via the de-repression of Cdkn2a. Since PRC2 insufficiency is often involved in the pathogenesis of MDS, the present results suggest that p53-dependent dyserythropoiesis manifests in MDS in the setting of PRC2 insufficiency.

3108 – MOLECULAR MECHANISMS OF LENALIDOMIDE RESISTANCE IN DEL(5Q) MYELODYSPLASTIC SYNDROMES

Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the commonest structural genomic variant in myelodysplastic syndromes (MDS). Lenalidomide (LEN) is the treatment of choice for patients with del(5q) MDS. Half of the responding patients become resistant within two years of which around 20% present TP53 mutations at the time of LEN resistance. To identify novel genetic determinants of LEN resistance, we compared protein coding variants from paired samples from 29 del(5q) patients who responded to LEN and eventually became resistant to treatment. Our analysis identified, in addition to TP53, recurrent RUNX1 alterations in relapsed patients. Exposure of del(5q) cell lines to LEN showed upregulation of RUNX1 protein levels and function in a CRBN and TP53 dependent manner. RUNX1 deletion, shRNA-mediated downregulation or RUNX1 mutant expression rendered sensitive cells unresponsive to LEN. LEN-induced IKZF1 degradation released the transcriptional activity of a RUNX1/GATA2 complex to drive megakaryocytic differentiation of del(5q) cells, which was required for Calpain-dependent del(5q) cell death. We confirmed these findings in human CD34+ cells expressing a shRNA targeting CSNK1A1 expression, which reproduce del(5q) cell LEN sensitivity. RUNX1 and TP53 loss-of-function in del(5q) cells blocked LEN induced megakaryocytic differentiation and subsequent cell death. Transcriptome analysis from paired del(5q) MDS patient samples showed loss of megakaryocytic gene signatures at relapse. Our results suggest that GATA2, RUNX1 and TP53 cooperate to drive megakaryocytic differentiation after LEN-mediated degradation of IKZF1. Mutations blocking LEN-induced megakaryocytic differentiation promote drug resistance and activating GATA2 may re-sensitize therapy-resistant del(5q) cells.