Mutations In Myelodysplastic Syndromes Research Articles

PARP inhibition leads to synthetic lethality with key splicing-factor mutations in myelodysplastic syndromes.

Splicing factors are frequently mutated in patients with myelodysplastic syndromes and acute myeloid leukaemia. Recent studies have revealed convergent molecular defects caused by splicing factor mutations, among which R-loop dysregulation and resultant genome instability are suggested as contributing factors to disease progression. On the other hand, understanding how mutant cells survive upon aberrant R-loop formation and genome instability is essential for developing novel therapeutics. The immunoprecipitation was performed to identify R-loops in association with PARP1/poly-ADP-ribosylation. The western blot, immunofluorescence, and flow cytometry assays were used to test the cell viability, cell cycle arrest, apoptosis, and ATM activation in mutant cells following the treatment of the PARP inhibitor. The Srsf2(P95H) knock-in murine hematopoietic cells and MLL-AF9 transformed leukaemia model were generated to investigate the potential of the PARP inhibitor as a therapy for haematological malignancies. The disease-causing mutations in SRSF2 activate PARP and elevate the overall poly-ADP-ribosylation levels of proteins in response to R-loop dysregulation. In accordance, mutant cells are more vulnerable to the PARP inhibitors in comparison to the wild-type counterpart. Notably, the synthetic lethality was further validated in the Srsf2(P95H) knock-in murine hematopoietic cell and MLL-AF9 leukaemia model. Our findings suggest that mutant cells antagonise the genome threat caused by R-loop disruption by PARP activation, thus making PARP targeting a promising therapeutic strategy for myeloid cancers with mutations in SRSF2.

A mitochondrial surveillance mechanism activated by SRSF2 mutations in hematologic malignancies.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

Abstract P26: Altered RNA Export Sensitizes to Nuclear Export Inhibition in SF3B1 Mutant MDS

Abstract SF3B1 mutations, the most frequent spliceosomal alterations across cancers, occur in 25% of myelodysplastic syndromes (MDS) patients yet lack effective therapies. Two phase 2 clinical trials with the XPO1 inhibitors in high-risk MDS revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs) out of the nucleus. Given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome, we hypothesized that XPO1 inhibition may preferentially affect SF3B1-mutant cells and that SF3B1-mutant high-risk MDS patients would have a better response to rational targeted drug combinations with XPO1 inhibitors. To evaluate the mechanism underlying the preferential sensitivity of SF3B1-mutant cells to XPO1 inhibition, we conducted subcellular RNA sequencing before and after XPO1 inhibition in SF3B1 wildtype and mutant cells. Transcriptomic analysis revealed increased global retention of RNA transcripts and elevated snRNAs in the nucleus after XPO1 inhibition in the SF3B1 mutant cell line. Global RNA expression and splicing analysis revealed increased alternative splicing in SF3B1 mutant cells after XPO1 inhibition. These results suggest that the preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through increased nuclear retention of spliceosomal snRNAs and select mRNAs that results in perturbation of apoptotic pathways. Despite the promising efficacy of XPO1 inhibition in SF3B1-mutated MDS, dose escalation is limited by toxicity. In order to identify novel drug combination targets with lower XPO1 inhibitor doses, we employed whole genome CRISPR screens in two acute myeloid leukemia (AML) cell lines treated with XPO1 inhibitor. We identified two drug targets that greatly synergized with eltanexor specifically in the SF3B1 mutant cell lines: venetoclax (a BCL2 inhibitor), and A1331852 (a BCL-XL inhibitor). In addition, BH3 profiling demonstrated increased priming of the BCL2 and BCL-XL in SF3B1 mutant cells. We further validated these combinations in vivo using competitive transplant assays in Sf3b1 K700E conditional knock-in mice. The combination of eltanexor with venetoclax and eltanexor with A1331852 showed a significant decrease in the Sf3b1-mutant burden in the peripheral blood and bone marrow progenitor compartments, suggesting a preferential sensitivity of the combinations for SF3B1 mutant cells. Although A1331852 exhibited similar results to venetoclax, there was increased weight loss and decrease in hemoglobin associated with BCLXL inhibition. In conclusion, our study provides insight on the mechanisms underlying the heightened sensitivity of XPO1 inhibition in SF3B1-mutant MDS/AML. The identification of BCL2 and BCL-XL as synergistic targets with XPO1 inhibitors, validated by in vivo testing, BH3 profiling, and RNA sequencing, highlights the potential for therapeutic combinations. Specifically, the combination of eltanexor and venetoclax could be a promising SF3B1-specific therapy. Citation Format: Sana Chaudhry, Felipe Beckedorff, Shaista Shabbir Jasdanwala, Tulasigeri M Totiger, Maurizio Affer, Nidhi Hariramani, Olivia Tonini, Stephan Noudali, Alexandra Chirino, Alyssa Cornista, Skye Montoya, Daniel Bilbao, Jumana Afaghani, Jose Antonio Rodríguez, Shruti Bhatt, Eric Wang, Justin Taylor. Altered RNA Export Sensitizes to Nuclear Export Inhibition in SF3B1 Mutant MDS [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P26.

SF3B1 K700E Alters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS

Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novo methyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1 and DNMT3A mutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3A mutations have shorter progression-free survival than those with solely SF3B1 mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood. To investigate how Dnmt3a and Sf3b1 mutations disrupt HSC function in vivo we crossed two conditional knock-in mouse models of common point mutations in SF3B1 (K700E) and DNMT3A (R787H, murine equivalent to human DNMT3A R882H). We followed monthly blood counts and evaluated stem and progenitor cell composition in the bone marrow at 6 months. We found that Sf3b1/Dnmt3a double mutant mice display a similar phenotype to Sf3b1K700E mice, with a progressive anemia and an expansion in LT-HSCs. However, when assayed for HSC self-renewal potential, whole bone marrow and LT-HSCs isolated from Sf3b1/Dnmt3a mutant mice display increased serial colony formation capacities similar to Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs, 5 to 6 passages), but have a competitive disadvantage in secondary and tertiary transplant assays that is less severe than Sf3b1K700E HSPCs but distinct from the competitive advantage of Dnmt3a mutant HSPCs. Transcriptionally, double mutant HSCs clustered closer to Sf3b1 HSCs with shared expression of 74% of upregulated genes and 64% of downregulated genes and have decreased expression of stress response and metabolism genes. These results indicate Sf3b1 mutations may mitigate the preleukemic phenotype due to Dnmt3a mutations in MDS. To identify factors regulated by SF3B1 mutations that may mitigate the effect of DNMT3A loss on leukemic progression, we generated isogenic DNMT3A KO and SF3B1 K700E single and double mutant K562 cell lines using CRISPR/Cas9. Transcriptome analysis demonstrated that SF3B1K700E/DNMT3AKO double mutant clones clustered with and shared similar upregulated (69%) and downregulated (62%) genes as SF3B1 single mutant clones. Similar to our findings in murine HSPCs, pathway analysis of SF3B1 and SF3B1/DNMT3A mutant clones displayed enrichment of stress response and metabolic pathways. Alternative mRNA splicing analysis identified 13 events (3 alternative 3' splice site, 9 intron retention, and 1 skipped exon) in 8 genes that were upregulated in DNMT3A KO clones and downregulated in SF3B1 and SF3B1/DNMT3A mutant clones. We confirmed decreased protein expression of these mis-spliced genes in SF3B1 single and double mutant clones, but not DNMT3A KO clones. These genes include components of the integrated stress response pathway, which are also mis-spliced in SF3B1 and SF3B1/DNMT3A mutant MDS patient samples. Taken together our data suggest that aberrant splicing by mutant SF3B1 prevents metabolic reprogramming and decreases leukemic progression caused by DNMT3A mutation in MDS.

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.

Interactions between Iron Overload, Oxidative Stress, and Somatic Mutations in Myelodysplastic Syndromes; Evidence from the Literature

Introduction: Somatic mutations (SM) are central to the pathophysiology of myelodyspastic syndromes (MDS). Iron overload (IOL) from transfusion of red blood cells (RBC) and the oxidative stress that results from the ability of iron to transfer electrons and undergo Fenton chemistry are common and are associated with adverse clinical endpoints (infections, cardiac events, exacerbation of bone marrow failure, event-free survival, progression-free survival, overall survival) in MDS. We reviewed the literature to elucidate what evidence is available indicating an interaction between iron, oxidative stress and specific SM in MDS. Methods: A series of PubMed searches were done including the key words of each specific mutation combined with iron, oxidative stress, and reactive oxygen species (ROS, a measure of oxidative stress), focusing on SM prevalent in MDS and/or included in the molecular International Prognostic Scoring System (IPSS-M; 31 mutations in all, including IPSS-M mutations and TET2). Citations relevant to hematopoietic stem and progenitor cells, cells of the hematopoietic microenvironment, and myeloid disorders were favoured for review. Mutations of familial predisposing conditions were also searched in combination with iron, oxidative stress and ROS. Results: Of 31 mutations found in the IPSS-M, an additional four mutations found in familial predisposing conditions (DDX41, GATA2, CHEK2, SAMD9) were searched as was TET2, for a total of 35 mutations. Fifty-four references were identified. Fifty-three references were preclinical/translational in nature, with one case report (WT1). The most frequent alterations in iron/ROS were with the SF3B1 mutation, with 11 citations. Thirteen of 31 (42%) of SM in combination with iron, oxidative stress/ROS had citations. Reports for combination with iron or oxidative stress/ROS, respectively, included early (TP53, IDH2, RUNX1, SF3B1, STAG2, TET2; and TP53, DNMT3A, IDH2, NPM1, U2AF1) and late (NRAS, CEBP3A; and CBL, NRAS) mutations. Both gain of function (TP53, IDH2, NRAS, SF3B1; and TP53, IDH2, NRAS, U2AF1) and loss of function (STAG2, TET2; and CBL) mutations had relevant citations identified. Cellular pathways affected included tumour suppressor (TP53 for both iron and oxidative stress/ROS), signal transduction (NRAS for both), splicing factors (SF3B1; U2AF1), DNA stability (STAG2) and (hypo) methylation (TET2; DNMT3A). There were reports of an impact at multiple levels on iron/ROS and cellular/systemic physiology in the context of SF3B1 mutation, for example: ERFE+12 (containing 12 additional nucleotides and translated to an ERFE protein containing 4 additional amino acids; 358 vs 354 in wild type) present in SF3B1 mutated MDS does like the usual ERFE suppress hepcidin, impacting iron at a systemic level; enzymes of iron, heme and iron sulfur cluster physiology are altered, impacting iron physiology at a cellular level; and mitochondrial ferritin sequesters iron, leading to cytoplasmic iron deficiency, lowering ROS and thus possibly protecting cells from oxidative damage. Table 1 shows examples of iron/ROS interactions with SF3B1, TET2, DNMT3A, U2AF1, STAG2, NRAS, TP53, IDH2, CBL, NPM, ETNK, PTPN11, WT1 and RUNX1. Figure 1 shows MDS pathophysiology and where interactions between SM and iron/oxidative stress/ROS may impact this pathophysiology. No investigations were identified relevant to the mutations of familial predisposing conditions, including DDX41, GATA2, CHEK2 and SAMD9 in combination with iron/oxidative stress/ROS. Discussion: Searching IOL, oxidative stress and ROS in combination with MDS somatic mutations uncovered information as to how iron/redox status may interact with SM to affect MDS pathophysiology. Of particular interest is the cytoplasmic iron deficiency induced in the presence of SF3B1 mutation, the only SM conferring favourable prognosis in MDS in the IPSS-M. Given the pleiotropic cellular, intracellular and subcellular mechanisms involved, investigation regarding when and how targeting iron overload by reducing RBC transfusion requirements, binding iron or attenuating ROS may alter cellular and clinical outcomes in the setting of specific somatic mutations is warranted.

Results of Phase I/II Study of Azacitidine in Combination with Quizartinib for Patients with Myelodysplastic Syndromes and Myelodysplastic/Myeloproliferative Neoplasms with FLT3 or CBL Mutations

Introduction: FMS-like tyrosine kinase 3 ( FLT3) mutations occur in approximately 1% of patients (pts) with newly diagnosed myelodysplastic syndrome (MDS) and up to 19% of pts at time of failure to hypomethylating agents (HMA). In addition, mutations in the Casitas B-lineage Lymphoma gene ( CBL) are observed in 10% of pts with MDS/MPN and up to 15% of pts with CMML. Preclinical studies suggest that CBL-mutant cells are dependent on FLT3 signaling. Here we present results of a phase I-II study of the FLT3 inhibitor quizartinib in combination with azacitidine for pts with FLT3 and CBL mutant MDS and MDS/MPN. Methods: We conducted a phase I/II clinical trial of azacitidine in combination with quizartinib for pts with MDS and MDS/MPN with detectable FLT3 and/or CBL mutations and with/without prior failure to HMA (HMA-F). The study included an initial phase I dose-escalation portion, following a 3+3 design, followed by a phase II dose expansion portion. Dose escalation included 3 dose levels of quizartinib: 30, 40 and 60 mg administered p.o daily on days 1-28 of each 28 day cycle. Therapy consisted on quizartinib (at corresponding dose level) in combination with azacitidine 75 mg/m 2/day i.v os s.c on days 1-5. The primary endpoint was to evaluate safety, tolerability and maximum tolerated dose of quizartinib. Responses were evaluated following 2006 IWG criteria. The Kaplan-Meier product-limit method was used to estimate median survival. Results: Between July 2020 and June 2023 a total of 16 pts have been treated: 12 in the phase I portion and 4 in the phase II. A total of 4 pts had MDS with excess blasts, 2 had MDS/MPN with neutrophilia and 10 had CMML. Eight (50%) pts had FLT3-ITD mutations and 8 (50%) had CBL mutations. Four (25%) pts had HMA-F one of which had received 4 lines of therapy including hematopoietic stem-cell transplant (HSCT) prior to enrollment. Based on the Molecular IPSS, 8 (50%), 3 (19%) and 5 (31%) pts had very high, high and moderate high risk disease. Among CMML pts, 3 (30%) pts had high and 7 (70%) had intermediate-2 risk by CPSS-Molecular score. In the phase I portion, 3 pts received quizartinib at dose level 1, 3 at dose level 2 and 6 at dose level 3. No dose limiting toxicities were detected during the 28-day DLT evaluation window. Dose level 2 was selected as the P2RD. Most common adverse events (AEs) were constipation (56%), fatigue (50%), insomnia (44%), anorexia (38%), cough (38%), diarrhea (38%) and arthralgia (31%). Most common grade 3-4 AEs were anemia (31%), thrombocytopenia (31%), lung infection (13%), skin infection (13%), hyponatremia (13%) and sepsis (13%). Arrythmias were observed 5 pts: atrial fibrillation (grade 2, n=2; grade 3, n=1), Mobitz type II atrioventricular block (grade 3, n=1), atrial flutter (grade 2, n=1), QTC prolongation (grade 2, n=1). The 4-week and 8-week cumulative incidences of mortality were 0%. Median number of days to cycle 2 was 30 (range 27-51). Median number of days to cycle 2 of therapy by dose level were: 30, 30 and 37 days for dose levels 1, 2 and 3, respectively (p=0.301). Dose reductions due to myelosuppression were required in 5 (31%) pts including dose reductions of azacitidine in 3 pts and of quizartinib in 3 pts. Median number of cycles was 4 (range 1-17). Median cycles to to best response was 1 (range 1-6). The overall response rate was 69% (n=11): CR in 1 (6%), mCR with HI in 2 (13%), mCR in 8 (50%) pts. FLT3 mutant pts were more likely to respond to therapy (100% vs 50%, p=0.038). Clearance (n=6, 75%) or allelic burden reductions (n=2, 25%) of FLT3-ITD mutations were observed in all FLT3 mutant pts. No clearance of CBL mutations were observed. Dynamics of absolute neutrophil count, platelets and hemoglobin during cycle 1 are shown in Figure 1A. Median response duration was 3.5 months (range 0-22 moths). With a median follow up of 19.1 months (95% CI 2.6-35.6), the median event-free survival has not been reached and the median overall survival is 17.5 months (NR vs 10.1 months in FLT3 vs CBL mutant pts, p=0.084, Figure 1B) Five (31%) pts discontinued study to proceed with HSCT at time of best response, 3 (19%) due to transformation to AML, 2 (12%) due to pt choice, 1 (6%) due to treating physician choice and 1 (6%) due to relapse. Four (25%) pts remain on study. Conclusion: Therapy with azacitidine in combination with quizartinib for pts with higher-risk MDS and MDS/MPN with FLT3 or CBL mutations has acceptable toxicity profile and is associated with promising responses mainly among FLT3-mutant pts.

E7820, an Anti-Cancer Sulfonamide, in Combination with Venetoclax in Patients with Splicing Factor Mutant Myeloid Malignancies: A Phase II Clinical Trial

*JPB and NSC contributed equally. Background and Significance: Mutations in RNA splicing factor genes ( SF3B1, SRSF2 U2AF1 and ZRSR2)are common in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). We and others have previously shown that pharmacologic degradation of the RNA splicing factor RBM39 preferentially kills splicing factor mutant cells. These preclinical data provided the rationale for a recently completed phase II clinical trial (NCT05024994) of the oral RBM39 degrader E7820 in patients with relapsed or refractory splicing factor mutant MDS or AML. The safety and efficacy data of this trial have been presented previously (Bewersdorf et al ASH 2022). The generally favorable safety profile and correlative studies confirming target engagement and induction of mis-splicing events support the further exploration of E7820 in the context of combination therapies. Recent clinical data show that treatment with venetoclax can overcome the negative prognostic impact of splicing factor mutations in AML (Senapati et al, Blood 2023). Additionally, preclinical data from our laboratory show synergy between E7820 and venetoclax in SF3B1-mutant isogenic cell lines leading to enhanced RBM39 degradation and blunting of MCL-1 expression, a common cause of venetoclax resistance ( Figure 1). Dynamic BH3 profiling of E7820 in myeloid leukemia cell lines suggests an E7820 dose-dependent priming for BAD which implies forced reliance on BCL2. Furthermore, adding venetoclax to E7820 further increased mitochondrial priming. Preclinical animal modeling with patient-derived xenografts (PDX) is ongoing. Based on these preclinical data, we are amending the study to include a separate arm of E7820 in combination with venetoclax. Methods and Study Design: This is an investigator-initiated, phase II trial (NCT05024994) that is being conducted at Memorial Sloan Kettering Cancer Center and Sylvester Comprehensive Cancer Center at the University of Miami of adult (≥18 years) patients with relapsed or refractory AML, MDS, or chronic myelomonocytic leukemia (CMML). Patients must have a hotspot splicing factor mutation in SF3B1, SRSF2, U2AF1 or U2AF2 (with hotspot mutations as defined by OncoKB) or a nonsense or frameshift mutation in ZRSR2 detected within 6 months of study screening for enrollment. Patients are required to have adequate end-organ function and an Eastern Cooperative Oncology Group (ECOG) performance status of ≤3. Prior treatment with venetoclax is permitted. Patients enrolled in the new combination arm will receive E7820 100mg by mouth once daily + venetoclax orally once daily on days 1-28 of cycle 1 on days 1-21 of subsequent cycles. The study design is shown in Figure 2. As this combination has never been studied in human AML and MDS patients previously, the study will include a 6-patient safety run-in phase with an optional dose de-escalation of E7820 to 70mg orally once daily. The sample size is based on a Minimax Simon 2-stage design. Assuming a type I error rate of 0.05, power of 0.8, a response rate of 10% in the historical control group and 30% with E7820 + venetoclax, 15 patients will be enrolled in stage 1. If there are at least 2 responses among the first 15 patients, we will enroll another 10 patients. The null hypothesis will be rejected if there are at least 6 responses in the 25 patients. The primary endpoint of this trial is to evaluate the efficacy of E7820 + venetoclax in patients with relapsed/refractory myeloid malignancies with mutations in splicing factor genes as measured by the response rate within 6 cycles of therapy (AML: complete remission (CR) + complete remission with partial hematologic recovery (CRh); MDS: CR + CRh + CR with limited hematologic recovery (CR-L) + partial remission (PR); CMML: CR + PR). Secondary endpoints include overall and event-free survival. Correlative biomarker and pharmacodynamic parameters will be assessed as exploratory endpoints including effects on RBM39 protein levels, changes in global and key target splicing events, and evaluation of DCAF15 mRNA levels and response to therapy.

STAG2 loss Induces HSC Programs By Modulating Accessibility of AP-1 Bound Enhancers

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are blood disorders characterized by clonal expansion of mutant hematopoietic stem and progenitor cells (HSPCs). Mutations in genes encoding the cohesin complex, including STAG2, RAD21, SMC3 and SMC1A, are frequent genetic drivers in MDS and AML. However, the mechanisms by which cohesin mutations lead to clonal expansion of HSPCs are not well understood. Cohesin is essential for sister chromatid cohesion, DNA damage repair, maintenance of chromatin architecture, and gene regulation. To investigate the impact of cohesin mutations on chromatin accessibility, we performed ATAC-Seq in a panel of six isogenic STAG2 wild type (WT) and knockout (KO) U937 leukemia cell lines. Among the total of 97,798 ATAC-Seq peaks, we identified significantly more STAG2 KO (8,859) versus WT (1,622) uniquely accessible chromatin sites, which were strongly enriched for intergenic regions. Integration of ChIP-Seq profiling of cohesin binding and histone modifications demonstrated cohesin binding and an increase in the active enhancer and promoter histone mark H3K27Ac in the STAG2 KO newly accessible chromatin, suggesting a direct role for cohesin in regulating the activity of such enhancers. Together, our data demonstrate that STAG2 KO-induced open chromatin may be enriched for novel STAG2 KO-specific enhancers. To further infer the regulatory function of these putative enhancers, we predicted binding of transcription factors (TFs) in ATAC-Seq-defined regions by performing TF footprinting using the Transcription factor Occupancy Prediction By Investigation of ATAC-seq Signal (TOBIAS) algorithm. We observed that binding of members of the AP-1 transcription family, including JUN FOS andATF, previously implicated in oncogenic transformation, was significantly enriched in the STAG2 KO cells (Figure 1A). Conversely, STAG2 KO cells lost CEBP TF binding in regions that lost chromatin accessibility. To identify potential regulatory targets of these TFs, we mapped TF footprints to annotated promoter regions and to enhancer-gene pairs determined by the Activity by Contact (ABC) model. We observed a strong correlation between predicted AP-1 binding and gene expression of putative regulated genes in STAG2 KO cells, further supporting the role of these newly accessible AP-1 bound sites as regulatory elements. To validate our findings using in vivo models of cohesin-mutant disease, we performed RNA-Seq and ATAC-Seq in HSPC isolated from a Tet2/Stag2 mouse model of MDS previously developed in our lab. We confirmed an increase in accessible chromatin and demonstrated a strong enrichment of predicted Ap-1 binding in intergenic regions, in agreement with our observations in the U937 model. Gene set enrichment analysis in the Tet2/Stag2-mutant versus WT HSPC revealed that putative targets of Ap-1 binding were enriched for hematopoietic stem cell gene signatures (Figure 1B) . This is consistent with our observation of clonal expansion of HSPCs in this model, and suggests a mechanism by which novel Ap-1 bound enhancers may facilitate expression of key genes involved in HSPC expansion and development of MDS/AML. To explore whether our findings were applicable to other published models of Stag2 mutant myeloid neoplasms, we performed TF footprinting using publicly available ATAC-Seq datasets generated in HSPC isolated from Stag2 and Runx1/Stag2 conditional knockout mice. We recapitulated increased chromatin accessibility and significant enrichment of cohesin-bound Ap-1 footprints in Stag2 mutant over WT conditions. Together, these data further validate conservation of STAG2 KO-induced AP-1 bound regulatory elements in human and mouse models of MDS and AML. In summary, we show that STAG2 loss leads to the establishment of a novel set of accessible intergenic regions with features of enhancer activity in vitro and in vivo. We identify multiple members of the AP-1 complex with enriched predicted binding at these putative enhancers, and demonstrate that AP-1 may drive expression of HSPC signatures in models of Stag2 mutant MDS in vivo. We illustrate here a potential role for the AP-1 complex in cohesin mutant myeloid malignancies, which we are currently functionally interrogating.

SF3B1 mutations in myelodysplastic syndromes: A potential therapeutic target for modulating the entire disease process.

Myelodysplastic syndromes (MDS) are clonal hematologic malignancies characterized by ineffective hematopoiesis and dysplasia of the myeloid cell lineage and are characterized by peripheral blood cytopenia and an increased risk of transformation to acute myeloid leukemia (AML). Approximately half of the patients with MDS have somatic mutations in the spliceosome gene. Splicing Factor 3B Subunit 1A (SF3B1), the most frequently occurring splicing factor mutation in MDS is significantly associated with the MDS-RS subtype. SF3B1 mutations are intimately involved in the MDS regulation of various pathophysiological processes, including impaired erythropoiesis, dysregulated iron metabolism homeostasis, hyperinflammatory features, and R-loop accumulation. In the fifth edition of the World Health Organization (WHO) classification criteria for MDS, MDS with SF3B1 mutations has been classified as an independent subtype, which plays a crucial role in identifying the disease phenotype, promoting tumor development, determining clinical features, and influencing tumor prognosis. Given that SF3B1 has demonstrated therapeutic vulnerability both in early MDS drivers and downstream events, therapy based on spliceosome-associated mutations is considered a novel strategy worth exploring in the future.

JAK2 Mutations Are Rare and Diverse in Myelodysplastic Syndromes: Case Series and Review of the Literature

To investigate and characterize JAK2 mutations in myelodysplastic syndrome (MDS), we present three cases with diverse JAK2 mutations and review the literature. The institutional SoftPath software was used to find MDS cases between January 2020 and April 2022. The cases with a diagnosis of a myelodysplastic/myeloproliferative overlap syndrome including MDS/MPN with ring sideroblasts and thrombocytosis were excluded. The cases with molecular data by next generation sequencing looking for gene aberrations commonly seen in myeloid neoplasms were reviewed for the detection of JAK2 mutations including variants. A literature review on the identification, characterization, and significance of JAK2 mutations in MDS was performed. Among 107 cases of the MDS reviewed, a JAK2 mutation was present in three cases, representing 2.8% of the overall cases. A JAK2 V617F mutation was found in one case representing slightly less than 1% of all the MDS cases. In addition, we found JAK2 R564L and JAK2 I670V point mutation variants to be associated with a myelodysplastic phenotype. JAK2 mutations in MDS are rare and represent less than 3% of cases. It appears that JAK2 variant mutations in MDS are diverse and further studies are needed to understand their role in the phenotype and prognosis of the disease.

Advances in the application of Raman spectroscopy in haematological tumours.

Hematologic malignancies are a diverse collection of cancers that affect the blood, bone marrow, and organs. They have a very unpredictable prognosis and recur after treatment. Leukemia, lymphoma, and myeloma are the most prevalent symptoms. Despite advancements in chemotherapy and supportive care, the incidence rate and mortality of patients with hematological malignancies remain high. Additionally, there are issues with the clinical diagnosis because several hematological malignancies lack defined, systematic diagnostic criteria. This work provided an overview of the fundamentals, benefits, and limitations of Raman spectroscopy and its use in hematological cancers. The alterations of trace substances can be recognized using Raman spectroscopy. High sensitivity, non-destructive, quick, real-time, and other attributes define it. Clinicians must promptly identify disorders and keep track of analytes in biological fluids. For instance, surface-enhanced Raman spectroscopy is employed in diagnosing gene mutations in myelodysplastic syndromes due to its high sensitivity and multiple detection benefits. Serum indicators for multiple myeloma have been routinely used for detection. The simultaneous observation of DNA strand modifications and the production of new molecular bonds by tip-enhanced Raman spectroscopy is of tremendous significance for diagnosing lymphoma and multiple myeloma with unidentified diagnostic criteria.

Splicing factor mutations in the myelodysplastic syndromes: Role of key aberrantly spliced genes in disease pathophysiology and treatment.

Mutations of splicing factor genes (including SF3B1, SRSF2, U2AF1 and ZRSR2) occur in more than half of all patients with myelodysplastic syndromes (MDS), a heterogeneous group of myeloid neoplasms. Splicing factor mutations lead to aberrant pre-mRNA splicing of many genes, some of which have been shown in functional studies to impact on hematopoiesis and to contribute to the MDS phenotype. This clearly demonstrates that impaired spliceosome function plays an important role in MDS pathophysiology. Recent studies that harnessed the power of induced pluripotent stem cell (iPSC) and CRISPR/Cas9 gene editing technologies to generate new iPSC-based models of splicing factor mutant MDS, have further illuminated the role of key downstream target genes. The aberrantly spliced genes and the dysregulated pathways associated with splicing factor mutations in MDS represent potential new therapeutic targets. Emerging data has shown that IRAK4 is aberrantly spliced in SF3B1 and U2AF1 mutant MDS, leading to hyperactivation of NF-κB signaling. Pharmacological inhibition of IRAK4 has shown efficacy in pre-clinical studies and in MDS clinical trials, with higher response rates in patients with splicing factor mutations. Our increasing knowledge of the effects of splicing factor mutations in MDS is leading to the development of new treatments that may benefit patients harboring these mutations.

Biological and clinical significance of TP53 mutations in myelodysplastic syndrome and acute myeloid leukemia

Anthracycline- and cytarabine-based intensive combination chemotherapies are considered the backbone therapy for patients with acute myeloid leukemia (AML). Although chemotherapy leads to long-term remission and cures many patients with AML, it can induce DNA damage/stress due to acute/chronic toxicities, acquired resistance, relapse, and therapy-related malignancies. Introduction of molecularly targeted agents with less systemic toxicities has considerably improved the scope of treatment, particularly in elderly and frail patients. However, outcomes of TP53-mutated myelodysplastic syndrome (MDS) and AML, a distinct group of myeloid disorders, have not improved irrespective of the treatment used (median overall survival, 5-10 months). In this review, we discuss the biological and clinical significance of TP53 mutations in malignancies, while particularly focusing on MDS/AML, and emerging therapies for TP53-mutated MDS/AML. Rationally designed novel treatment strategies are expected to improve the clinical outcomes of TP53-mutated MDS/AML.

SF3B1, RUNX1 and TP53 Mutations Significantly Impact the Outcome of Patients With Lower-Risk Myelodysplastic Syndrome.

Prognosis of patients with myelodysplastic syndrome (MDS), particularly the group with lower-risk disease (LR-MDS) is very heterogeneous. Several studies have described the prognostic value of recurrent somatic mutations in MDS including all risk categories. Recently, the incorporation of genomic data to clinical parameters defined the new Molecular International Prognostic Scoring System (IPSS-M). In this study, we evaluated the impact of molecular profile in a series of 181 patients with LR-MDS and non-proliferative chronic myelomonocytic leukemia. Epigenetic regulators (TET2, ASXL1) and splicing (SF3B1) were the most recurrent mutated pathways. In univariate analysis, RUNX1 or TP53 mutations correlated with lower median overall survival (OS). In contrast, SF3B1 mutation was associated with prolonged median OS [95 months (95% IC, 32-157) vs. 33 months (95% CI, 19-46) in unmutated patients (P < 0.01)]. In a multivariate Cox regression model, RUNX1 mutations independently associated with shorter OS, while SF3B1 mutation retained its favorable impact on outcome (HR: 0.24, 95% CI, 0.1-0.5; P = 0.001). In addition, TP53 or RUNX1 mutations were identified as predictive covariates for the probability of leukemic progression (P < 0.001). Incorporation of molecular testing in LR-MDS identified a subset of patients with expected poorer outcome, either due to lower survival or probability of leukemic progression.

Distinct genetic landscapes and their clinical implications in younger and older patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clinically and genetically diverse diseases that impose patients with an increased risk of leukemic transformation. While MDS is a disease of the elderly, the interplay between aging and molecular profiles is not fully understood, especially in the Asian population. Thus, we compared the genetic landscape between younger and older patients in a cohort of 698 patients with primary MDS to advance our understanding of the distinct pathogenesis and different survival impacts of gene mutations in MDS according to age. We found that the average mutation number was higher in the older patients than younger ones. The younger patients had more WT1 and CBL mutations, but less mutated ASXL1, DNMT3A, TET2, SF3B1, SRSF2, STAG2, and TP53 than the older patients. In multivariable survival analysis, RUNX1 mutations with higher variant allele frequency (VAF) and U2AF1 and TP53 mutations were independent poor prognostic indicators in the younger patients, whereas DNMT3A and IDH2 mutations with higher VAF and TP53 mutations conferred inferior outcomes in the older patients. In conclusion, we demonstrated the distinct genetic landscape between younger and older patients with MDS and suggested that mutations impact survival in an age-depended manner.

Incidence, Clinical Associations, and Co-Mutation Patterns of UBA1 Mutations in MDS

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). However, the prevalence and characteristics of UBA1 mutations in MDS are unknown. We hypothesize that UBA1 mutations underlie a proportion of MDS cases with systemic inflammation. Methods Molecular data were reviewed from a representative diagnostic and treatment-naive MDS cohort (n= 3,328) ascertained through the International Working Group for MDS (Bernard E et al NEJM Evid 2022). We prioritized 375 cases for UBA1 genotyping based on: 1) male gender; and 2) no identified driver mutations or few mutations, enriched in DTA (DNMT3A, TET2, ASXL1) genes; or 3) unclassifiable (MDS-U or MDS/MPN-U) per 2016 WHO Classification. We identified UBA1 hotspot mutations (M41T/V/L) with digital droplet PCR. Resulting UBA1 status was integrated with cytogenetic and sequencing data from 121 genes involved in myeloid pathogenesis to identify patterns of co-mutation. Clinical associations of UBA1 mutations were evaluated and where available, clinical history was reviewed for inflammatory conditions. Results We identified 30 UBA1 M41T/V/L mutations (median number mutant droplets: 1301; range: 4-12166) in 8% of the cohort (n=28 of 375 patients). This included 15 patients with M41T (c.122T>C), 7 with M41V (c.121A>G), 4 with M41L (c.121A>C), and 2 patients with more than one mutation (1 with M41V/T, and 1 with M41L/V). In 79% of UBA1-mutant cases (22/28), the karyotype was normal. Loss of the Y chromosome was observed in 6 cases. Three cases had MDS-associated cytogenetic abnormalities: del(11q), del(13q) and focal del(20q). In 43% of UBA1-mutant cases (12/28), UBA1 mutations were the sole genetic abnormalities. For a further 43% we observed co-mutation with DTA genes (DNMT3A, TET2, ASXL1) with median variant allele fraction (VAF) of 0.21 (0.02-0.56). SF3B1 co-mutations with VAF >30% were observed in 3 cases. Most UBA1-mutant cases were classified per 2016 WHO as MDS-SLD/MLD (n=18), but two cases were MDS with excess blasts 1, and one case was CMML-1 with 9% blasts. The median age at diagnosis was 72 years (44-89). No significant difference was found when comparing clinical parameters (blast counts, complete blood counts, age) between UBA1 mutant and wildtype MDS MDS. Clinical history was readily available for 4 UBA1-mutant patients. Inflammatory symptoms were reported in 3 patients, localized in the lungs (n=2), skin (n=2) and cartilage (n=1). Two patients also had a diagnosis of inflammatory/rheumatological disease (vasculitis and relapsing polychondritis), two patients had thrombosis, one had a history of monoclonal gammopathy of undetermined significance. The 4th patient, without inflammatory symptoms, had a prior diagnosis of T-cell large granular lymphocytic leukemia. Follow-up for transformation to acute myeloid leukemia (AML) and overall survival was available for 310 and 339 patients, respectively. No UBA1-mutant patients transformed to AML (n=24; median follow-up 2.6 years). There was no difference in overall survival between UBA1-mutant (n=27) and wildtype (n=312) patients (log-rank test p = 0.7), however, when subsetting for patients with no mutations detected by targeted DNA sequencing (UBA1-mutant n=7; wildtype n=45), UBA1-mutant patients had worse overall survival (median 5.2 vs 7.3 years; log-rank test p = 0.03). ConclusionUBA1 mutation in isolation or in combination with other mutations implicated in myeloid pathogenesis account for a significant proportion (8%) of patients diagnosed and treated as MDS but without established disease classification. Ongoing work includes 1) clinical review of inflammatory symptoms for the remaining UBA1-mutant cases and 2) profiling a larger cross-section of MDS by a UBA1 targeted panel to characterize mutations beyond the M41 locus.

Synthetic Data Generation By Artificial Intelligence to Accelerate Translational Research and Precision Medicine in Hematological Malignancies

Background In hematological malignancies there is a growing demand for real-world, comprehensive data including clinical and genomic information to build powerful models to improve diagnosis, prognosis and personalized treatment choice. However, collecting such information in large patient populations is challenging and there are many issues concerning patient privacy that need to be accounted for. One approach that can circumvent these issues is the creation of synthetic data that captures the complexities of the original data set (distributions, non-linear relationships, and noise) without including any real patient information. Aims 1) Apply advanced synthetic data generation methods to real-world datasets of different hematological malignancies. 2) Develop a Synthetic Validation Framework to evaluate the quality of synthetic data and perform data augmentation. 3) Test the capability of synthetic data to accelerate translational research. Methods Here we implemented a Conditional Tabular Wasserstein Generative Adversarial Networks (GAN) architecture with Gradient Penalty to generate synthetic data. Use cases were different cohorts of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) with available clinical and molecular features. We created a Synthetic Validation Framework to evaluate the quality of generated synthetic data: Clinical Synthetic Fitness (CSF) and Genomic Synthetic Fitness (GSF) scores were calculated as the average of multiple metric tests adopted. Patients were stratified by Hierarchical Dirichlet (HD) clustering. Explainability analysis was carried out by SHapley Additive exPlanations approach (SHAP). Survival analyses were performed by Kaplan-Meier curves and CoxPH models (Experimental plan is reported in Figure 1). Results We first created a synthetic copy of a MDS cohort (n=2,043) using all the real data for training the model. We compared synthetic vs. real data, obtaining high fitness performances for both clinical and genomic features (CSF=93%; GSF=90%). HD were then applied to define clusters capturing broad dependencies among genomic features, showing comparable results in synthetic vs. real data; SHAP analysis indicated that similar features drive patients’ classification in both datasets. Finally, synthetic patients had comparable survival with respect to real ones; when applying conventional scoring system (IPSS-R), the probability of survival of the 5 risk categories was comparable between synthetic and real data. In the second experiment setting, we analysed synthetic MDS datasets with different size generated with model trained on a real dataset. Interestingly, when generating a synthetic augmented dataset (200%) we obtained high fitness performance for both clinical and genomic features (CSF=91%; GSF=89%). Moreover, all the performances showed a similar trend when considering a cohort of 1,002 patients with AML (CSF=92%; GSF=89%) thus proving evidence for high generalizability of the model across different clinical settings. Finally, we investigated if the generation of synthetic data can accelerate translational research in hematology. Since the first publication on clinical relevance of gene mutations in MDS (Leukemia 2014;28:241), it took several years to collect data in large patient populations for generating a molecular classification (JCO 2021;39:1223) and prognostic score (IPSS-M, NEJM Evid 2022;1:7). Starting from the MDS cohort available in 2014 (n=944, Leukemia 2014;28:241), we generated 300% augmented synthetic dataset. HD were applied to synthetic data to define genomic-based clinical entities, resulting in the identification of the same 8 subgroups described in a real cohort of 2,043 patients many years later. Moreover, we applied a CoxPH model to the synthetic dataset to generate a molecular prognostic score (IPSS-M_Syn). The model was based on similar molecular features as the real IPSS-M and identified 6 risk categories in which the probability of survival was similar to that of IPSS-M risk groups (Figure 2). Conclusion GAN-generated synthetic data recapitulate statistical properties and complexity of clinical and genomic features in different hematological malignancies, replicate reliable survival estimates and allow effective data augmentation. The implementation of this technology seems to accelerate precision medicine research in hematology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Impact of Monoallelic Versus Biallelic TP53 Alterations in Intensively-Treated Adults AML Patients: A Retrospective Study from the ALFA Group

Introduction:TP53 is a key tumor suppressor gene involved in apoptotic pathways, metabolism functions, preservation of genomic balance and DNA repairs. TP53 locus is subject to mutations, deletions and copy-neural loss of heterozygosity. TP53 mutations are associated with a reduced overall survival and are assigned to the ELN 2017 adverse-risk group. Recent studies have suggested a worse prognostic impact of biallelic TP53 alterations compared to monoallelic TP53 mutations in myelodysplastic syndromes (Bernard et al, Nat. Medicine, 2020). Whether TP53 allelic state impairs the prognostic in intensively-treated acute myeloid leukemia (AML) patients and may refine the treatment strategy has not been addressed. Here, we analysed TP53 alterations in newly diagnosed AML patients (pts) from 3 prospective Acute Leukemia French Association (ALFA) trials. Methods: A total of 983 adult AML pts enrolled in ALFA-0701 (NCT00927498), ALFA-0702 (NCT00932412) and ALFA-1200 (NCT01966497) trials were retrospectively screened for TP53 alterations by high throughput sequencing and conventional cytogenetics. Pts displaying at least two TP53 mutations or one TP53 mutation associated with either TP53 locus deletion or TP53 locus copy-neutral loss of heterozygosity (CN-LOH) were assigned as biallelic TP53 alteration patients. TP53 locus CN-LOH were approximated based on variant allele frequencies and copy number analysis. Survival was analysed with multivariate Cox models stratified on the trial. Results: Eighty-three patients had TP53 mutations (8%; ALFA-0701: n=21, ALFA-0702: n=28; ALFA-1200: n=34), including 14 pts with monoallelic and 69 pts with biallelic TP53 alterations respectively. Median age of TP53 mutated patients was 62 years (IQR: 57-68). Leukocytes counts and bone marrow blast counts at diagnosis were lower in TP53 mutated patients (3.0 G/L vs. 8.7 G/L, p<0.0001; 40% vs. 58%, p=0.002, respectively). Median variant allele frequency of TP53 mutations was 43% (IQR: 21-69). As compared to TP53 wildtype AML, TP53 mutated pts more often harbored complex karyotype (n=69 (86%) vs n=51 (6%), p<0.0001) and monosomal karyotype (n=65 (81%) vs n=32 (4%), p<0.0001). The median number of additional mutations was 2 (IQR: 0-3). TP53 mutations were associated with mutations in signalling genes (NF1, NRAS, PTPN11, KRAS, FLT3, KIT and JAK2) in 33 pts (40%). Overall, 43 TP53 mutated AML patients (52%) achieved complete remission (CR)/complete remission without platelet recovery (CRp) after one or two courses of induction whereas 684 TP53-wt patients (76%) were in CR/CRp at that time (p<0.0001). TP53 mutations were associated with a significantly reduced OS and DFS in multivariate analysis including age and WBC count (2-year OS, 11% vs 56%, HR: 3.48, 95% CI: 2.71-4.47, p<0.0001; 2-year DFS, 12% vs 50%, HR: 2.97, 95% CI: 2.12-4.16, p<0.0001, respectively). Among TP53 mutated AML, biallelic TP53 alterations were associated with worse OS compared to monoallelic TP53 alterations (2-year OS, 4% vs 43%, HR: 4.13, 95% CI: 1.87-9.13, p=0.0004 in multivariate analysis accounting for age and WBC count). Interestingly, outcome of the 2017 ELN risk stratification intermediate group was not different to that of monoallelic TP53 pts (2-year OS, 43% for monoallelic TP53 pts vs 53%, HR: 1.07, 95% CI: 0.55-2.07, p=0.84). In pts with biallelic TP53 alterations, presence of signalling mutations was associated with a worse survival in a multivariate analysis accounting for age and WBC (HR: 1.82, 95% CI: 1.06-3.11, p=0.03). Conclusion: Allelic status refines the prognostic value of TP53 mutations in AML. While monoallelic TP53 alterations display similar outcome to that of the ELN 2017 intermediate risk group, biallelic TP53 alterations dramatically impair patient outcome. Patients with biallelic TP53 alterations may be good candidates for new first-line treatment strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combined Structural Variant and Mutation Profiling for Comprehensive Characterization of Chromoanagenesis and TP53 Mutations in Myelodysplastic Syndromes with Complex Karyotype

Combined Structural Variant and Mutation Profiling for Comprehensive Characterization of Chromoanagenesis and TP53 Mutations in Myelodysplastic Syndromes with Complex Karyotype