Chronic airway eosinophilia is associated with allergic asthma and is mediated in part by secretion of IL-5 from allergen-specific Th2 lymphocytes. IL-5 is a known maturation and antiapoptotic factor for eosinophils and stimulates release of nascent eosinophils from bone marrow into the peripheral circulation. An antisense oligonucleotide found to specifically inhibit IL-5 expression in vitro was observed to significantly reduce experimentally induced eosinophilia in vivo, in both the murine OVA lung challenge and allergic peritonitis models. Intravenous administration resulted in sequence-dependent inhibition of eosinophilia coincident with reduction of IL-5 protein levels, supporting an antisense mechanism of action. Potent suppression of lung eosinophilia was observed up to 17 days after cessation of oligonucleotide dosing, indicating achievement of prolonged protection with this strategy. Furthermore, sequence-specific, antisense oligonucleotide-mediated inhibition of Ag-mediated late phase airway hyperresponsiveness was also observed. These data underscore the potential utility of an antisense approach targeting IL-5 for the treatment of asthma and eosinophilic diseases.

Single-stranded oligodeoxynucleotides (ODNs) were tested for their ability to stimulate NK cells isolated from murine spleens to lyse target cells. Various sequences were evaluated, some of which have been shown previously to exhibit pharmacologic activity in murine model systems. It was confirmed that the CpG motif was stimulatory only in specific sequence contexts, and we found that phosphorothioate backbones were, in general, less stimulatory than phosphodiester backbones. In addition, this stimulation could be reduced by methylating the cytosine of the CpG and eliminated by modifying all of the cytosines contained in an ODN with methyl, bromo, or iodo modifications to the 5 position of the cytosine ring. These results were compared with the ability of a subset of these ODN sequences to stimulate B cell proliferation in vitro. In this comparison, phosphorothioate backbones were found to be required, and the context of the CpG motif was found to be less critical for activation. Finally, one of the most potent ODNs was shown to activate NK and B lymphocytes when administered in vivo.

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3´splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.

Introduction: TP53 mutant (mTP53) MDS and AML represent a distinct molecular cohort with poor outcomes. Hypomethylating agents (HMA) have emerged as preferred treatment for these patients with CR rate of 20-30% and median OS of 6-12 months. APR-246 is a mutant p53 activator with single agent activity in mTP53 AML. We report initial phase 1b results of APR-246 + AZA in mTP53 MDS/AML. Methods: Eligible pts included HMA naïve mTP53 MDS and oligoblastic AML (≤ 30% blasts) ≥ 18 years of age. Pts received APR-246 in a 3+3 dose escalation design (50, 75, 100 mg/kg lean body weight) 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. Primary objective was safety with AEs graded by CTCAE v4.03 and DLT assessment over 6 weeks. Secondary endpoints 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. Results: As of Jan 1, 2017, 9 pts (33% male; median age 65 years (39-73)) have enrolled with 3 pts per cohort. Three pts had AML-MRC and 6 had MDS; all pts had poor risk cytogenetics (11% poor, 89% very poor) and higher risk disease by IPSS-R (22% high, 78% very high). Median BM blasts were 18% (4-30). Seven pts (78%) remain on study: 2 pts in the 50mg/kg cohort discontinued treatment (Tx), 1 pt due to infection during C2 who later died of sepsis unrelated to Tx, and 1 pt electively discontinued in durable marrow CR (mCR) after 5 cycles of therapy. Median time on study is 106 days (14-221). Tx related AEs during the lead-in phase (all G1) included ataxia (n=1), dizziness (n=1), and facial numbness (n=1). AEs occurring in > 1 pt included dizziness (n=3), nausea (n=3), neutropenia (n=3), thrombocytopenia (n=3), infection (n=3), headache (n=2), pain (n=2), weakness (n=2), falls (n=2), facial numbness (n=2), and ataxia (n=2); all G1/G2 except neutropenia/thrombocytopenia (G4). No Tx-related SAEs or DLTs have occurred to date. Five of six pts were response evaluable with 1 pt discontinuing tx prior to 1st disease assessment. ORR by IWG was 100% with 4 CR (80%, 3/3 in DL2) and 1 mCR. All CR patients achieved complete cytogenetic response. One CR patient achieved a 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 (55-70%) which normalized on serial assessment (<5%). Serial NGS with a variant allele frequency (VAF) cutoff of 2% was negative in 80% of patients (4/5). The remaining 3 pts entered the study in Dec 17/Jan 18 with no response data available at data cutoff. Conclusions: APR-246 + AZA combination is well tolerated in mTP53 MDS/AML. Responses have been achieved in all pts (80% CR) accompanied by deep molecular remissions. The maximum tolerated dose has not been reached and dose escalation is ongoing. Citation Format: David A. Sallman, Amy DeZern, Kendra Sweet, David P. Steensma, Thomas Cluzeau, Mikkael Sekeres, Guillermo Garcia-Manero, Gail Roboz, Amy McLemore, Kathy McGraw, John Puskas, Ling Zhang, Chirag Bhagat, Armin Graber, Najla H. Al Ali, Eric Padron, Roger Tell, Jeffrey E. Lancet, Pierre Fenaux, Alan F. List, Rami S. Komrokji. Phase Ib/II combination study of APR-246 and azacitidine (AZA) in patients with TP53 mutant myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT068.

Background: Only a small subset of Lower risk (LR) MDS patients benefit from treatment with rhu-Erythropoietin (Epo). We previously reported that lenalidomide (LEN) restores sensitivity to Epo in MDS progenitors by inducing the formation of lipid rafts that are enriched for signaling competent, JAK2/Epo-receptor complexes (McGraw K, et. al. PLoS One 2014; Basiorka A, et. al. Cancer Res 2016). In the MDS-002 and MDS-005 trials, treatment with LEN monotherapy gave rise to RBC transfusion-independence (TI) in 26% of azanucleoside-naïve, transfusion-dependent (TD) LR, non-del(5q) MDS patients for a median of 10.2 and 7.75 months, respectively. In a pilot study of Epo-refractory LR-MDS patients, the addition of epoetin alfa (EA) to LEN treatment yielded erythroid responses in 28% of patients who were unresponsive to LEN alone, suggesting that LEN may overcome clinical resistance to augment response to rhEpo (Komrokji R, et. al. Blood 2012). To test this hypothesis, we performed a randomized phase III trial comparing treatment with LEN to LEN+EA in LR non-del(5q) MDS patients who were refractory to, or not candidates for treatment with rhEpo. Methods: Patients with Low or Intermediate-1 IPSS risk MDS with hemoglobin <9.5 g/dL who were unresponsive to rhEpo treatment or were TD (>2 units/month) with serum Epo >500mU/mL were eligible. Patients were stratified by serum Epo level and prior rhEpo (EA vs. darbepoetin vs. none) then randomized to treatment with LEN 10 mg/d x21d q4wk (Arm A) or LEN + EA 60,000U SC/wk (Arm B). The primary endpoint was major erythroid response (MER) at week 16 which was defined according to transfusion status at baseline: (1) achievement of RBC-TI for ≥ 8 consecutive weeks AND a sustained ≥1 g/dL hemoglobin rise compared to mean pre-transfusion baseline value in TD patients; and (2) a >2 g/dL rise in hemoglobin without transfusion for ≥ 8 consecutive weeks in non-TD patients (<4U RBC/8 wks). Arm A non-responders were offered cross-over to combined therapy. Results: Among 205 patients randomized, 14 were excluded from the primary analysis due to a 4 month interruption in drug supply. Among the 195 evaluable patients, 96 were assigned to Arm A and 99 to Arm B. Baseline characteristics were well balanced between arms with 85% of patients heavily TD, receiving a median of 4 units/8 weeks. Overall, 93% of patients received prior treatment with Epo and 18% azanucleosides. In an intent to treat analysis, 28/99 patients (28.3%) in Arm B achieved MER compared to 11/96 (11.5%) in Arm A (P=0.004). Among 136 patients who completed 16 weeks of study treatment, 28/72 (38.9%) and 10/64 (15.6%) achieved MER, respectively (P=0.004). Forty-four Arm A non-responders crossed over to combination-therapy with 11 patients (25%) experiencing a MER. Multivariable logistic regression analysis identified only treatment with LEN + EA as an independent covariate for erythroid response (P=0.01). Responses were durable with MER median duration of 23.8 months in Arm B compared to 13 months in Arm A. There was no significant difference in the frequency or distribution of >Grade 3, non-hematologic adverse events between treatment arms. Two patients progressed to AML while on study (Arm A), and no thromboembolic events were reported. Conclusions: LEN restores sensitivity to rhEpo in otherwise refractory, LR-non-del(5q) MDS patients to yield a significantly higher frequency of durable major erythroid responses compared to LEN alone. The addition of LEN to EA treatment is an effective strategy for the management of Epo-refractory patients with a potential duration of benefit extending to years. Disclosures List: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Verma:Janssen: Research Funding; BMS: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Komrokji:JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; celgene: Consultancy; pfizer: Consultancy. Luger:Onconova: Research Funding; Pfizer: Honoraria; Seattle Genetics: Research Funding; Cyslacel: Research Funding; Biosight: Research Funding; Ariad: Research Funding; Agios: Honoraria; Genetech: Research Funding; Jazz: Honoraria; Daichi Sankyo: Honoraria; Kura: Research Funding; Celgene: Research Funding. Mattison:Pfizer: Membership on an entity's Board of Directors or advisory committees. Altman:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy, Honoraria, Other: Data Safety and Monitoring Committee; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; France Foundation: Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; prIME Oncology: Speakers Bureau; PeerView: Speakers Bureau; Cancer Expert Now: Consultancy; Novartis: Consultancy; Biosight: Other: US Lead. Claxton:Astellas Pharma: Other: Pharma support of clinical studies; Merck Sharp & Dohme Corp.: Other: Pharma support of clinical studies; Cyclacel Pharmaceuticals, Inc.: Other: Pharma support of clinical studies; Medimmune Inc.: Other: Pharma support of clinical studies; Novartis Pharmaceuticals: Other: Pharma support of clinical studies; Celgene Corporation: Other: Pharma support of clinical studies; Incyte Corporation: Other: Cyclacel Pharmaceuticals, Inc; Daiichi Sankyo Co. and Ambit Biosciences Corp: Other: Pharma support of clinical studies. Artz:Miltenyi: Research Funding. Tallman:Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Lenalidomide used for treatment non-del 5q myelodysplastic syndromes.