[Notice to Mrs. Martha Parks, 26 July 1907]

An official notice of the allowance of the claim for the Widow's Pension of Mrs. Martha Parks from Milo B. Stevens & Co

Cancer stem cells, epithelial-mesenchymal transition, ATP and their roles in drug resistance in cancer

The cancer stem cell (CSC) state and epithelial-mesenchymal transition (EMT) activation are tightly interconnected. Cancer cells that acquire the EMT/CSC phenotype are equipped with adaptive metabolic changes to maintain low reactive oxygen species levels and stemness, enhanced drug transporters, anti-apoptotic machinery and DNA repair system. Factors present in the tumor microenvironment such as hypoxia and the communication with non-cancer stromal cells also promote cancer cells to enter the EMT/CSC state and display related resistance. ATP, particularly the high levels of intratumoral extracellular ATP functioning through both signaling pathways and ATP internalization, induces and regulates EMT and CSC. The three of them work together to enhance drug resistance. New findings in each of these factors will help us explore deeper into mechanisms of drug resistance and suggest new resistance-associated markers and therapeutic targets

Hotspot U2AF1 Mutations Determine Missplicing Selectivity: Novel Mechanisms Altering Splicing Factors

Mutations in splicing factor genes (SF3B1, SRSF2, U2AF1, and ZRSR2) are identified in over 50% of patients diagnosed with myelodysplastic syndrome (MDS). U2AF1 is a U2 auxiliary factor that forms a heterodimer with U2AF2 for the recognition of the 3' splice site (SS) and results in the subsequent recruitment of U2snRNPs during pre mRNA splicing. U2AF1 mutations are present in 11% of MDS and its presence is correlated with an increased risk of progression to AML. Non-canonical mutations are rarely seen in U2AF1 but two highly conserved hotspots (S34, Q157) are frequently seen and result in distinct downstream effects. We performed NGS analysis of a large cohort of patients with myeloid neoplasms (MN; n=3,328) and identified 238 patients with U2AF1 mutations (7%). We analyzed the somatic mutational profile, clonal hierarchy, and splicing profile of patients with U2AF1S34 (n=99), U2AF1Q157 (n=119), and others (n=20; M1/*, A26T/V, R35Q, R118C, E124K, F150L, E152G, C154S, R156H, M172L). The mutational spectrum of U2AF1S34 and U2AF1Q157 was equally dismal but pretty distinct. U2AF1 S34 cases were mostly co-mutated for DNMT3A (5%), TET2 (4%), RUNX1 (2%), ASXL1, CBL, ETV6, KRAS, NRAS (1.3%), STAG2 (1%), CUX1, and TP53 (<1%) while U2AF1Q157 had higher numbers of mutations in ASXL1 (10%), SETBP1, TET2 (3%), NRAS (2%), DNMT3A, PHF6 (2%), JAK2 (2%), CBL, EZH2, TP53 (2%), IDH2, RUNX1, STAG2 (2%), KRAS (1%), and IDH1 (<1%). Mutational rank showed: U2AF1S34 was ancestral in 38% of the cases followed by secondary DNMT3A and NRAS (5%, both). Ancestral U2AF1Q157 was found in 35%, with ASXL1 (19%) being the most common secondary hit. Subclonal U2AF1S34 (44%) was most commonly preceded by DNMT3A (21%), while secondary U2AF1Q157 (40%) had ASXL1 (28%) as the most common first hit. U2AF1S34 and U2AF1Q157 were co-dominant to a miscellanea of mutations. U2AF1S34 cases had a shorter OS than U2AF1Q157 cases (n=82 vs. n=101; 20 vs. 25 mo.; P=.002). Ancestral U2AF1S34 or U2AF1Q157 defined a dismal prognosis compared to secondary U2AF1S34 or U2AF1Q157 (OS: n=63 vs. n=86; 29 vs. 37 mo.; P=.03). To investigate the effects of both mutations on splicing patterns, we analyzed RNA-Seq profiles, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events that were classified as skipping exon (SE), retained intron (RI), and 3' or 5' alternative SS (A3SS, A5SS) (Hershberger, ASH 2019). Overall, 675 AS events in 430 genes were scored significant (pFDR<.05) when the splicing inclusion/exclusion difference was ±10%. U2AF1S34 and U2AF1Q157 caused an equal fraction of SE (79% U2AF1S34; 72% U2AF1Q157). Only 4% of the genes were commonly misspliced by both mutations, while the rest of the genes were uniquely spliced according to each mutation. Some exemplary genes misspliced by both mutations were DDX3X (an RNA helicase) showing a consistent SE at exon 5 and an RI 3-5a and CCNG1 (cell growth regulation) which was enriched for RI 7-7. Among others, U2AF1S34 uniquely affected the splicing of TET2 (SE e3); cell cycle regulators, CDC37L1 (SE), CCNC (SE e8), and HDAC3 (SE e6). We then investigated whether U2AF1 mutations might affect the splicing of other RNA splicing components. This mechanism would lead to the loss of regulation of the spliceosome complex. U2AF1S34 produces selective RIs in SRSF2 (4-4a) and A5SS in SRSF6 (7a and 7). Tumor suppressors and proto-oncogenes were also found to be misspliced by U2AF1S34 including PTEN (RI 3-Ua), CTNNB1 (RI 15-19; 3'SS (19 and 19a), and CCNL1 (RI 4-U). Major regulators of splicing factor activity are phosphatases like PP1R12A and PPP1R12B, which showed an RI 8-7 and an A3SS, respectively. Among genes exclusively misspliced by U2AF1Q157, we identified DEAD-Box helicases [DDX17 (RI), DDX59 (SE e8), DHX29 (RI)], ALAS family members (ALAS1, SE e6; ALAS2, SE e5) and UTX (KDM6A; SE e16). U2AF1Q157 affected the missplicing of DYRK1A (SE e7), a kinase known to phosphorylate SF3B1 at T434 site. In sum, our study suggests that while concurrent mutations in splicing factors lead to lethality, the presence of mutations (as the case of U2AF1S34 and U2AF1Q157) and consequent missplicing of other splicing factors are permissive events in MN and might represent novel mechanisms of disease pathogenesis. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nazha:Abbvie: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Novartis: Speakers Bureau; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy

Can Monosomy 7 be Targeted By Next Generation Cereblon-Modulating Agents?

Genomic advances have initiated a new era of precision oncology. Targeted therapeutics can rationally select potentially responsive patient populations and exclude resistant patients based on molecular mechanisms of action. The application of immunomodulatory drugs (IMiDs) evolved from unselective usage to the identification of del(5q) as a target, which then led to mechanistic clues and clarification of its mode of action. Lenalidomide (LEN) has been used in non del(5q) patients with a variable response rate, but the molecular underpinnings for LEN efficacy in this setting are not known. Genomic analyses (Negoro et al., Leukemia 2016) have provided only a marginal improvement in response prediction, suggesting that other factors might be relevant in sensitizing cells to LEN. The effects of LEN are related to its binding affinity to cereblon (CRBN), the substrate adaptor of the CRL4CRBN E3 ubiquitin ligase, a cullin-ring ligase consisting of damaged DNA-binding protein1(DDB1), cullin4a (CUL4A), and regulator of cullins-1 (ROC1). LEN selectively activates CRL4CRBN E3 ubiquitin ligase to selectively degrade CSNK1A1 leading to synthetic lethality in patients with del(5q) that are haploinsufficient (HI) for CSNK1A1. However, LEN and the new generation compounds pomalidomide and iberomide (CC-220) also trigger recruitment and degradation of the zinc finger transcription factors IKAROS (IKZF1, 7p12.2) and AIOLOS (IKZF3, 17p12-q21.1) through CRBN, and therefore promote their proteosomal degradation. The synthetic lethality of these immunomodulator agents relies on expression levels of AIOLOS and IKAROS. Response to those agents depends strictly on low expressions of AIOLOS and IKAROS sensitizing certain cell types (e.g., myeloma). We hypothesized that levels of LEN sensitivity might correlate with expression levels of its targets in selected disease subtypes of myeloid neoplasia (MN). We studied a large cohort of patients with MN (n=3,328) and we identified 122 patients with -7 and 194 patients with del(7q). We noted that for those cases with informative RNA-seq expression, IKAROS exhibited haploinsufficient (HI) mRNA levels in 67% of patients with -7 and 40% in del(7q), as defined based on levels <25%tile of the diploid expression of cases (Kerr, ASH 2019). Since IKAROS is described to be a target of iberomide, we hypothesized that monosomy 7 (-7) but not del(7q) could selectively benefit from this agent, analogous to LEN in del(5q). Consistent with this proposition we further selected 160 hematopoietic cancer cells with available IKZF1 CNV status in a publicly available database of cell drug sensitivity (cancerrx). For all cell lines, the anti-proliferative effect of LEN was described. Cells with monosomy of IKZF1 (CNV=1) had an increased sensitivity to LEN as their IC50 was lower than compared to that of cells with IKZF1 with CNV ≥ 2 (6.1μM vs. 24.3μM). To further validate this prediction, we revisited the responsiveness to LEN (defined by Negoro et al., Leukemia 2016) of non-del(5q) MDS patients (n=79) showing an ORR of 40%. By focusing on patients with alterations in chr7 we identified 10 patients with MDS and high or very high International Prognosis Scoring System-Revised (IPSS-R) risk [6 patients with -7 and 4 patients with del(7q)]. In this cohort LEN response was assessed at 3 and 6 months; 4 out 6 patients with -7 responded to LEN, while del(7q) patients were all refractory (n=4). Responding monosomy 7 patients with complete bone marrow response (n=3) received 10 or 50mg doses of LEN (2 patients had a complex karyotype and 1 had isolated -7). The remaining responding patient had a partial response with hematological improvement. Interestingly, this patients was treated with a 5mg dose of LEN and had a TP53 mutation. In the other 2 patients: one with -7, LEN was discontinued because of pancytopenia associated with treatment, while in the other one -7 could not be confirmed by FISH. In the group of del(7q) patients, complex karyotypes and the presence of del(5q) in 2 did not predict for better response. Our preliminary results suggest that selective application of new generation IMiDS targeting IKAROS have clinical potential in patients with -7. Supportive data from in vitro cellular models will be comprehensively presented. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nazha:Tolero, Karyopharma: Honoraria; Incyte: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmacutical: Research Funding; Daiichi Sankyo: Consultancy; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy

RORA Is a Potential Prognostic Biomarker and Therapeutic Target for Patients with Acute Myeloid Leukemia

One strategy to improve precision medicine in acute myeloid leukemia (AML) is to further our understanding of the biological factors that influence pharmacologic efficacy. In a recent study, whole transcriptome analysis was conducted using pre- and post-treatment samples from a cohort of relapsed or refractory (R/R) AML patients treated with mitoxantrone, etoposide, cytarabine (MEC), and ixazomib. Logistic regression and linear discriminant analysis identified RORa as a predictor of response to treatment (Advani, et al., Clin Cancer Res, 2019, 4231-4237). RORa is not frequently mutated in AML but is described to be a tumor suppressor in patients with solid tumors. In the latter patient population, increased RORa expression is also associated with improved survival. We first retrospectively evaluated the prognostic significance of RORa in a larger cohort of R/R patients with AML (BeatAML). Second, we characterized RORa expression in cellular models of AML and sought to determine whether a commercially available RORa/RORγ agonist, SR1078, has anti-proliferative capacity in leukemia cell lines. For the correlative survival analyses, RORa mRNA expression and clinical information was downloaded from the BeatAML cohort (Tyner, et al., Nature, 2018, 526-531). Patients were categorized into high expressers (≥ median) and low expressers (<median). In total, 121 R/R patients were investigated. The mean age of diagnosis was 62 ± 12 years, and 58% of patients were male. The most common specific diagnosis at inclusion was AML with myelodysplasia related changes (28%), followed by AML with mutated NPM1 (18%) and therapy-related myeloid dysplasia (13%). Over half of the analyzed samples were peripheral blood (58%), and the remaining samples were either bone marrow aspirate (58%) or from leukapharesis (2%). As a whole, the median mRNA expression levels of RORa in patients with R/R AML (n=121) compared to healthy subjects (n=21) were 6.6 log2 CPM vs. 2.5 log2 CPM (P<.0001). After grouping patients into low RORa expresser and high RORa expresser groups, patients with above median expression of RORa were found to have significantly longer overall survival (19 mo. vs. 13 mo.; P=.0052; Figure 1). During normal hematopoiesis, we observed that RORa expression decreased with the stages of cellular maturation and is highly expressed in cells of AML patients with complex karyotype (BloodSpot, 2018). We noted that RORa mRNA expression varied across patients suggesting differences between AML subtypes. Expression levels were also confirmed to be different across AML subtypes in vitro by using cell line models (K-562, KG-1, OCI-AML3, U-937, THP-1, MOLM-13). In particular, analysis of TCGA data showed higher mean RORa expression in DNMT3A mutant (MT) (626 RPKM) compared to mean RORa expression in CEBPAMT (243 RPKM; P=0.026), NPM1MT (167 RPKM; P=0.012), and FLT3MT (236 RPKM; P=.003) AML patients. We then analyzed the sensitivity of cell lines to the commercially available synthetic RORa /RORγ ligand, SR1078. Myeloid lineage cells U-937 and KG-1 were used as a cellular model. Cells in logarithmic phase were treated with increased concentrations of the RORa /RORγ agonist SR1078 (from 3 nM to 30 mM) for 24 hours. Cell viability was measured by MTT tetrazolium reduction assay. Maximal growth inhibition was reached at 30 µM for U-937 (80%) and KG-1 (75%), respectively. We then combined SR1078 with MEC to evaluate whether in vitro SR1078 increased MEC growth inhibition. The addition of SR1078 to MEC significantly decreased cell viability in KG-1 cells compared to MEC alone (82% vs. 19%, P=.029). Our study suggests that increased RORa expression may be associated with improved survival in patients with R/R AML and that RORa may be a potential therapeutic target in AML. Figure 1 Disclosures Gerds: Celgene Corporation: Consultancy, Research Funding; Imago Biosciences: Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Roche: Research Funding; Sierra Oncology: Research Funding. Mukherjee:Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Projects in Knowledge: Honoraria. Nazha:Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; Daiichi Sankyo: Consultancy. Maciejewski:Alexion: Consultancy; Novartis: Consultancy. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Advani:Abbvie: Research Funding; Pfizer: Honoraria, Research Funding; Glycomimetics: Consultancy, Research Funding; Amgen: Research Funding; Macrogenics: Research Funding; Kite Pharmaceuticals: Consultancy

Rare germline alterations of myeloperoxidase predispose to myeloid neoplasms

Myeloperoxidase (MPO) gene alterations with variable clinical penetrance have been found in hereditary MPO deficiency, but their leukemia association in patients and carriers has not been established. Germline MPO alterations were found to be significantly enriched in myeloid neoplasms: 28 pathogenic/likely pathogenic variants were identified in 100 patients. The most common alterations were c.2031-2 A &gt; C, R569W, M519fs* and Y173C accounting for about half of the cases. While functional experiments showed that the marrow stem cell pool of Mpo(-/-) mice was not increased, using competitive repopulation demonstrated that Mpo(-/-) grafts gained growth advantage over MPO wild type cells. This finding also correlated with increased clonogenic potential after serial replating in the setting of H2O2-induced oxidative stress. Furthermore, we demonstrated that H2O2-induced DNA damage and activation of error-prone DNA repair may result in secondary genetic damage potentially predisposing to leukemia leukemic evolution. In conclusion, our study for the first time demonstrates that germline MPO variants may constitute risk alleles for MN evolution