ALBERT

Description: We previously reported that S100A9 promotes ineffective hematopoiesis and the development of MDS in a feed forward age-dependent fashion. Nonetheless, the precise mechanism by which S100A9 may foster DNA damage in MDS remains unclear. We recently showed that S100A9 directs overexpression of the fat-mass and obesity-associated gene (FTO) encoding an m6A RNA demethylase, which leads to nuclear exclusion of SRSF2. Removal of SRSF2 from its functional domain in the nucleosome leads to stalling of RNA polymerase II and formation of the nucleic acid R-loops, comprising DNA:RNA hybrids with the associated non-template single-stranded DNA. S100A9/FTO axis activation leads to SRSF2 deregulation through suppression of its main nuclear transport protein RanBP2, thereby stalling transcription machinery with resulting accumulation of nuclear R-loops and cytosolic/extracellular RNA:DNA hybrids. Persistent R-loops induce DNA damage while also compromising DNA repair. Here we identify an S100A9/FTO-regulated pathway responsible for induction of genomic instability through the accumulation of cytoplasmic RNA:DNA hybrids and modification of the spliceosomal patterns of aged S100A9Tg mice matching MDS hematopoietic stem and progenitor cells (HSPC). We first investigated which components of the S100A9/FTO axis are critical to hematopoiesis and those that are important for both the development of RNA:DNA hybrids and γH2AX activation. We analyzed the contribution of RanBP2 and the effects of elimination of R-loop formation via overexpression of RNAse H1, an enzyme that removes stalled R-loops in the nucleus by degrading DNA-hybridized RNA, thereby reducing the accumulation of cytoplasmic RNA:DNA hybrids. CRISPR knock-down of RanBP2 showed that the protein is critical for accumulation of yH2AX defined by double stranded breaks (DSB). Importantly, overexpression of RNAse H1 degraded R-loops and restored colony-forming capacity, indicating that RNA:DNA hybrids induced by the S100A9/FTO have profound effects on hematopoietic potential. However, while the FTO exclusion of SRSF2 from the nucleus explains the accumulation of γH2AX, it should potentially impact global RNA splicing. To investigate this, we performed a comparative RNAseq analysis on WT and S100A9Tg mice (young and old) to understand both changes induced through the normal aging process as well as those compounded by S100A9. We found that genes linked to splicing, RNA development, nucleotide excision repair and genomic instability and ribosome function were downregulated in aged S100A9Tg mice. Further analysis comparing splicing patterns of S100A9Tg and WT mice with human MDS BM HSPC led to ~200 common genes that were analyzed further. These genes showed that there are splicing changes enriched in spliceosomal assembly and mRNA splice selection site genes. We also found that the enriched genes affect the nucleolus and ribosome formation matching what is seen phenotypically with MDS. Dysregulation of these pathways are highly consistent with our observations of the pathways affected by the S100A9/FTO-induced inflammaging process, validating our hypothesis of S100A9 as a common initiator of dysfunction that can give rise to MDS. Importantly, our data demonstrates the potential for spliceosomal dysfunction regardless of the presence of spliceosomal mutations in MDS. We are currently in the process of performing both DRIPseq and m6A-seq of primary human MDS specimens and S100A9Tg mice to further assess the role of the S100A9/FTO pathway in the selection of sites for RNA/DNA hybrid formation and rise of genomic dysfunction that gives rise to MDS. We conclude that S100A9/FTO-induced nuclear exclusion of SRSF2 aids in the formation of RNA:DNA-hybrids that lead to genomic instability and the disruption of normal spliceosomal patterns in both human HSPC and the S100A9Tg MDS murine model, representing a previously uncharacterized mechanism contributing to MDS pathogenesis. Our studies provide evidence that targeting this cascade offers significant potential for development of novel, biologically rational therapeutics for MDS. Disclosures List: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction: TP53 gene mutations (mTP53), found in up to 20% of MDS or AML pts and 30-40% of therapy-related (TR) MDS/AML cases, represent a distinct molecular cohort with poor outcomes. Hypomethylating agents (HMA) are the standard of care with CR rates of ~20% and median OS of 7-8 months. APR-246 is a novel, first-in-class small molecule that selectively induces apoptosis in mTP53 cancer cells via thermodynamic stabilization of the p53 protein and shifting equilibrium toward the wild-type conformation. We previously reported the Phase 1b results of APR-246+AZA with no DLTs, transcriptional activation of p53 targets and high response rates, identifying a Phase 2 (P2) dose of 4500mg days 1-4 (Sallman et al., ASH 2018). We report herein the planned, completed phase 2 results. Methods: This is a multicenter Phase 1b/2 trial of APR-246+AZA in HMA-naïve mTP53 higher risk MDS, MDS/MPN and oligoblastic AML (≤ 30% blasts) pts (NCT03072043). P2 pts received APR-246 4500mg IV (days 1-4) + AZA 75 mg/m2 SC/IV x 7 days (days 4-10 or 4-5 and 8-12) in 28 day cycles. Primary objective was CR rate by International Working Group (IWG) 2006 criteria. Secondary objectives included ORR, OS, outcome following allogeneic hematopoietic stem cell transplant (allo-HSCT), and both next generation sequencing (NGS) and p53 immunohistochemistry (IHC) to monitor clonal suppression and remission depth as prognostic covariates. For minimal residual disease (MRD) analysis, a custom target-capture NGS assay was developed using unique molecular Identifiers for error correction with a 0.1% limit of detection. Results: As of July 15, 2019, 55 pts were enrolled (6 P1; 49 P2) with a median age 66 years (34-85; 47% male). By WHO, 40 pts had MDS, 11 AML-MRC and 4 CMML/MDS-MPN; 85% had complex cytogenetics and 33% TR-MDS/AML. All pts had higher risk disease by IPSS-R (7% Intermediate, 24% High, 69% Very High). Fifty pts (91%) had a TP53 missense mutation in the DNA binding domain with multiple mutations in 18 (33%), and median variant allele frequency (VAF) of 25%. In 34 pts (62%), TP53 was the sole mutation. Median time on treatment is 154 days (11-392) with 8 pts ongoing. Eighteen pts (33%; 40% of evaluable pts) discontinued study treatment to proceed to allo-HSCT. Treatment (Tx)-related AEs in ≥ 20% of pts included nausea/vomiting (58%), dizziness (31%), constipation (24%), neuropathy (22%), leukopenia (22%) and thrombocytopenia (20%; all G1/G2 except cytopenias (G3/G4). Tx-related febrile neutropenia and anemia occurred in 9% and 5% of pts with no other G3/G4 event in 〉1 pt. Thirty and 60 day mortality was 2% (n=1) and 6% (n=3), respectively. At data cutoff, 45pts were response evaluable with a median follow up of 10.5 months (Fig 1A). ORR by IWG was 87% (39/45) with 24 CR (53%), 8 marrow CR (mCR)+HI (18%), 3 HI alone (7%), and 4 with mCR (9%). Of 6 non-responders, 4 had stable disease and 2 pts had progressive disease. Median time to response was 2.1 months (0.1-5.4) and median duration of response of 6.5 months. CR rate for MDS was 61% (20/33), 50% for AML (4/8) and 0% for MDS/MPN (0/4) with an 88% ORR rate for MDS/AML and 75% for MDS/MPN. An isolated mTP53 was predictive for a higher CR rate (69% vs 25%; P=.006) with a trend for higher ORR (93% vs 75%; P=.17). Additionally, pts with 〉10% p53 IHC+ BM-MNC was a covariate associated with higher CR rate (66% vs 13%; P=.01). Complete and partial cytogenetic response occurred in 41% (n=18) and 18% (n=8) of pts, respectively. On serial TP53 NGS using a VAF cutoff of 5%, 39% (n=21) of patients achieved NGS negativity, which was associated with improved OS (12.8 vs 9.2 months; P=.02). In NGS- pts, the median MRD VAF at maximum clearance was 0.63% (0.0%-5%) with 5 pts (11%) MRD negative. By intention-to-treat analysis, median OS was 11.6 months (95% CI 9.2-14) with significantly longer OS in responding pts (12.8 vs 3.9 months; P

Description: Introduction: TP53 mutant (mTP53) MDS and AML, accounting for 5-10% of de novo MDS and 25-30% of therapy related MDS (t-MDS), represent a distinct molecular cohort with inferior outcomes. Hypomethylating agents (HMA) are preferred treatments for patients (pts) with these mutations, although with CR rates of only 20-30% and median OS of 6-12 months. APR-246 is a novel, first-in-class small molecule that selectively induces apoptosis in mTP53 cancer cells through mutant p53 protein re-activation by restoring the wild-type conformation, with single agent activity in mTP53 AML. We report the planned, completed Phase 1b results of APR-246+ azacitidine (AZA) in mTP53 MDS/AML. Methods: This is a multicenter Phase 1b/2 trial of APR-246+AZA in HMA naïve mTP53 MDS and oligoblastic AML (≤ 30% blasts) pts ≥ 18 years of age. Pts received APR-246 in a 3+3 dose escalation design (50, 75, 100 mg/kg lean body weight (equivalent to 4500mg fixed dose based on PK studies)) IV daily over 4 days in a lead-in phase (days -14 to -10) followed by the same dose of APR-246 (days 1-4) + AZA 75 mg/m2 SC/IV over 7 days (days 4-10 or 4-5 and 8-12) in 28 day cycles. The primary objective was to define safety and the recommended Phase 2 dose (RP2D), with AEs graded by CTCAE v4.03 and DLT assessment over 6 weeks. Secondary objectives included response by IWG 2006 criteria as well as serial next generation sequencing (NGS) and p53 IHC for evaluation of clonal suppression and remission depth as predictors of outcomes. For minimal residual disease (MRD) analysis, a custom target-capture NGS assay was developed using unique molecular Identifiers for error correction with a limit of detection of 0.1% with results validated by pt specific digital droplet PCR (ddPCR). Nanostring nCounter RNA expression analysis was conducted on a panel of 770 genes after the lead-in phase to assess transcriptional effects induced by APR-246. Results: As of July 30, 2018, 12 pts (42% male; median age 66 years (39-73)) were enrolled. Three pts had AML-MRC and 9 had MDS; all pts had poor risk cytogenetics (17% poor, 83% very poor) and higher risk disease by IPSS-R (25% high, 75% very high). T-MDS occurred in 5 pts (42%) and 7 pts (58%) were transfusion-dependent at baseline. Median BM blasts were 9% (4-30). Eleven of 12 pts (92%) had a TP53 missense mutation in the DNA binding domain with multiple mutations in 4/12 pts (33%). For 9/12 pts (75%), TP53 was the sole mutation. Median time on study is 176 days (41-298) with 7 pts ongoing. Treatment (Tx) related AEs during the lead-in phase (all G1) included nausea (n=5), neuropathy (n=5), decreased appetite (n=2), and dizziness (n=2) which were all transient. Tx related AEs occurring in 〉 1 pt in the combination phase included nausea/vomiting (n=6), dizziness (n=3), headache (n=3), neuropathy (n=3), fall (n=2), pruritus (n=2), thrombocytopenia (n=6), neutropenia (n=5), and leukopenia (n=4); all G1/G2 except cytopenias (G3/G4). No DLTs have occurred to date. Eleven of twelve pts were response evaluable with 1 pt discontinuing tx prior to 1st disease assessment (Fig 1A). ORR by IWG was 100% (11/11) with 9 CR (82%) and 2 marrow CR (mCR; 18%). Median time to first response was 70 days (4-91) and one CR patient achieved mCR and partial cytogenetic response after APR-246 lead-in prior to combination therapy. All CR pts had high p53 positivity by IHC at baseline (25-80%) which normalized on serial assessment with the 2mCR pts having

Description: Somatic gene mutations are key determinants of outcome in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). In particular, patients with TP53 mutations represent a distinct molecular cohort with uniformly poor prognosis. The precise pathogenetic mechanisms underlying these inferior outcomes have not been delineated. Here we characterize the immunological features of the malignant clone and alterations in the immune microenvironment in TP53 mutant and wild type MDS and sAML patients. Notably, PDL1 expression is significantly increased in hematopoietic stem cells of TP53 mutant patients, which is associated with MYC upregulation and marked down-regulation of MYC's negative regulator miR-34a, a p53 transcription target. Notably, TP53 mutant patients display significantly reduced numbers of bone marrow infiltrating OX40+ cytotoxic T-cells and helper T-cells, as well as decreased ICOS+ and 4-1BB+ NK cells. Further, highly immunosuppressive regulatory T-cells (i.e., ICOSHigh/PD-1neg) and MDSCs (PD-1low) are expanded in TP53 mutant cases. Finally, a higher proportion of bone marrow infiltrating ICOSHigh/PD-1neg Tregs is a highly significant independent predictor of overall survival. We conclude the microenvironment of TP53 mutant MDS and sAML has an immune privileged, evasive phenotype that may be a primary driver of poor outcomes, and submit that immunomodulatory therapeutic strategies may offer a benefit for this molecularly-defined subpopulation.

Description: Myelodysplastic Syndromes (MDS) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, Nlrp3 inflammasome (IFM) driven pyroptotic cell death and the induction of interferon-stimulated genes (ISG). Toll-like receptor 9 (TLR9) is an endosomal, DNA sensing pattern recognition receptor that primes and activates the IFM and ISG response through myddosome signaling upon engagement by hypomethylated, CpG-rich DNA. Oxidized newly synthesized mitochondrial DNA (ox-mtDNA) is released into the cytosol upon TLR/IL-1R activation to trigger Nlrp3 IFM activation. Upon lytic pyroptotic cell death, however, ox-mtDNA is released into the extracellular space. We previously reported that concentrations of ox-mtDNA, a native TLR9 ligand, are profoundly increased in MDS patient plasma compared to age-matched controls and other hematologic malignancies (Ward G, et. al. ASH 2018). The aim of this investigation was to determine if ox-mtDNA acts as a danger associated molecular pattern (DAMP) to propagate the inflammatory response and IFM activation in neighboring cells through TLR9. We have shown that MDS hematopoietic stem and progenitor cells (HSPC) redistribute TLR9 to display cell surface TLR9 expression. We hypothesized that this increased surface expression is induced in response to ox-mtDNA in the BM plasma. To test this, SKM1 and U937 cells were incubated for 2 hours with 50ng/mL ox-mtDNA (ND1 gene, unmethylated, amplified with oxidized guanosine) and TLR9 expression was assessed by flow cytometry (FC). Following incubation, both cell lines significantly increased TLR9 surface expression (n=3, p

Description: Background: The pathogenesis of Myelodysplastic Syndromes (MDS) is linked to constitutive innate immune stimulation that converges upon the NLRP3 inflammasome to induce pyroptosis, a caspase-1 dependent cell death. We have shown that inflammasome assembly is initiated by both cell-extrinsic stimuli such as S100A9 elaborated by Myeloid-Derived Suppressor Cells (MDSC), as well as cell-intrinsic somatic gene mutations (SGM) (Basiorka A, et. al. Blood 2016). SGM of varied classes evoke replication stress caused by transcriptional pauses that can expose genomic DNA to cytosolic sensors through unresolved R-loops or micronuclei formation. The cGMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) is a cell-intrinsic DNA surveillance pathway recognizing both cytosolic pathogenic and autologous DNA, leading to interferon stimulated gene (ISG) transcription and NLRP3 inflammasome activation, key biological features of MDS (Pellagatti A, et. al. Blood 2006; 108:337.). Here, we investigate the contribution of genomic cytosolic DNA engagement by cGAS-STING to NLRP3 inflammasome activation in MDS. Methods: MDS patient and healthy donor bone marrow mononuclear cells (BMMC) were isolated by Ficoll®-Hipaque method from consented participants at the Moffitt Cancer Center or the National Taiwan University Hospital (NTUH). Immortalized murine C57BL/6 Tet2-/- and MX1Cre/SRSF2P95H as well as respective wild type (WT) control BMMCs were used as MDS SGM models. Results: We first assessed cGAS-STING activation in MDS BMMC by measuring ISG response by microarray, demonstrating profoundly increased expression of ISG15, CXCL10, Samd9l, and Ifi27l2 in MDS BMMC (n=213) compared to healthy control BMMC (n=20) (p

Description: Constitutive innate immune activation is a pathogenetic driver of Myelodysplastic Syndromes (MDS) that directs ineffective hematopoiesis by NLRP3 inflammasome (IFM) assembly and pyroptotic cell death. IFM activation involves recruitment of caspase-1 (casp1) through the adapter protein, ASC, to facilitate autocatalytic cleavage of the zymogen to its active form that is responsible for interleukin (IL)-1β maturation, membrane pore formation and pyroptosis. Oxidized mitochondrial DNA (ox-mtDNA) has been proposed to serve as an alarmin that can activate the IFM by interaction directly with NLRP3 or engagement by DNA sensors, Toll-like receptor 9 (TLR9) and Cyclic GMP-AMP synthase (cGAS). Upon cytolysis, ox-mtDNA is released, permitting interaction with pattern recognition receptors on neighboring cells (Grishman, Pediatric Research, 2012, Shimada, 2012, Immunity. Vollmer, 2004, Immunology). Here, we investigate ox-mtDNA as an IFM-activator and pyroptotic biomarker in MDS. Incubation of TLR9 expressing cell lines, SKM1 (high expresser) and U937 (moderate expresser) with 50ng/mL ox-mtDNA (ND1 gene, amplified with oxidized guanosine) induced IFM activation evidenced by increased p-NFkβ, casp1 and IL-1β cleavage, ASC oligomerization and liberation of ASC specks. Direct interaction of ox-mtDNA with NLRP3 was confirmed by NLRP3 immunoprecipitation followed by probing for mtDNA using ND1 and CYTB specific primers and GAPDH primers as negative genomic control; mtDNA oxidation status was confirmed by dot blot. Furthermore, significantly increased expression of interferon stimulated genes (ISG) was seen in MDS bone marrow (BM) specimens (p≤0.01) compared to normal donors indicating TLR9 and/or cGAS activation. Ox-mtDNA engagement of TLR9 and cGAS was confirmed in MDS specimens by IF colocalization with corresponding IFM activation, as well as in MDS somatic gene mutation murine models (Tet2, SRSF2, U2AF) vs. Wt controls. Evaluation of surface TLR9 by flow cytometry showed significantly increased membrane expression in MDS CD34+ BMMC (n=4) vs. healthy donors (n=13) (p