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Background: Navitoclax (Nav) is a small-molecule inhibitor of several antiapoptotic B-cell lymphoma 2 (BCL2) family proteins, including BCL-xL, BCL2, and BCL-W. Preclinical studies have demonstrated that when administered alone, Nav can effectively induce apoptosis in various types of cancerous cells, including those isolated from patients (pts) with myeloproliferative neoplasms (MPNs). Ruxolitinib (Rux), a potent Janus kinase 1/2 inhibitor, can durably improve symptoms, splenomegaly, and overall survival in pts with myelofibrosis (MF) and is approved for the treatment of pts with primary MF in many countries, including Japan and Taiwan. However, pts can develop resistance to Rux, which may be overcome by the addition of Nav to a combination therapy regimen. An ongoing phase 2 study (NCT03222609) is assessing the safety and efficacy of Nav + Rux combination therapy in pts with MF, and a phase 1 study (NCT04041050) is evaluating the safety and pharmacokinetics (PK) of Nav monotherapy in Japanese pts with MPNs in part 1 and Nav + Rux combination therapy in Japanese and Taiwanese pts with MF in part 2. Herein we report the currently available results of this phase 1 study, the first to assess Nav treatment in Japanese and Taiwanese pts with MPNs including MF, polycythemia vera (PV), or essential thrombocythemia (ET). Methods: This phase 1, multicenter, open-label study enrolled pts ≥20 years of age with diagnosis of MPNs: MF, PV, or ET per World Health Organization classification. Eligible pts for part 1 of this study had an Eastern Cooperative Oncology Group performance status of 0 or 1 and were unable to tolerate/declined standard therapy or had MPNs that were unresponsive to standard therapy. Pts received Nav monotherapy at a starting dose of 50 mg orally once per day (QD) with a stepwise dose increase every ≥7 days to a maximum of 300 mg QD on the basis of tolerability. Per protocol, pts could receive treatment until disease progression, unacceptable toxicity, or the end of clinical benefit. Primary safety objectives included assessing the number of pts with dose-limiting toxicities (DLTs) within 28 days of Nav initiation and the number of pts with adverse events (AEs) throughout the study. DLTs included: grade (G)≥2 bleeding associated with low platelet counts of any G; bleeding requiring platelet transfusion; unexpected G≥2 toxicity requiring dose modification or delay of ≥1 week attributable to Nav; and all other G≥3 AEs considered related to Nav (exceptions: G3/4 leukopenia, G3 neutropenia, G4 neutropenia Results: As of May 2020, 6 female Japanese pts (median age: 62.5 years [range 41-79]) were enrolled in part 1 of this phase 1 study and diagnosed with the following: post-PV MF (n=1), post-ET MF (n=1), PV (n=2), and ET (n=2). All pts had received ≥1 prior therapy. Safety data are presented in the Table. The treatment duration for all 6 pts was between 4.6-24.7 weeks as of this cutoff, and all 6 pts were able to receive the maximum 300-mg dose of Nav, with no DLTs reported in any pts. No pts experienced a G≥4 AE during this period. Only 1 of 6 pts experienced a serious AE (SAE); this pt was discontinued and removed from the study on day 52 due to an SAE of epilepsy considered unrelated to Nav treatment. Five of 6 pts experienced a drug-related AE that led to study drug interruption and dose reduction, and all 5 pts remained on study and continued treatment. The current/most recent dose of Nav for all pts (excluding the pt who discontinued) was between 200-300 mg QD. No deaths were reported. Conclusions: Nav monotherapy had a tolerable safety profile in Japanese pts with MPNs enrolled in part 1 of this phase 1 study, with no DLTs reported. No clinically meaningful differences in safety were detected in this study compared with previous Nav monotherapy studies for hematologic malignancies. Preliminary PK and efficacy analyses for Part 1 are in progress, and Part 2 is ongoing with results to be reported in the presentation. Disclosures Shibayama: Celgene, Chugai, Eisai, AstraZeneca: Consultancy; Celgene, Ono, Takeda, AbbVie, Eisai, Novartis, Janssen, Chugai, Astellas, Teijin, MSD, Shionogi, Sumitomo Dainippon, Taiho, Nippon Shinyaku: Research Funding; Takeda, Novartis, Janssen, Ono, Chugai, Eisai, Kyowa Kirin, Sumitomo Dainippon, AstraZeneca, AbbVie, Daiichi Sankyo, Fujimoto, Nippon Shinyaku, Sanofi, Bristol-Myers Squibb, Pfizer, Otsuka, Mundipharma: Honoraria. Tanaka:Novartis Pharma KK, Celgene K.K: Speakers Bureau. Yeh:Amgen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees. Hsiao:AbbVie: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Nuthalapati:AbbVie: Current Employment, Other: may own stock. Ono:AbbVie: Current Employment, Other: may own stock. Uehara:AbbVie: Current Employment, Other: may own stock. Okubo:AbbVie: Current Employment, Other: may own stock. Komatsu:Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding.

Studies in large-scale genome sequencing have shown that only 2% of the mammalian genome encodes mRNAs, but the most part is transcribed as long and short non-coding RNAs (ncRNAs). The ncRNAs with gene regulatory functions are starting to be seen as a common feature of mammalian gene regulation. Genomic imprinting is a form of epigenetic regulation and imprinted genes are silenced in a parental-specific manner. Although the exact mechanism how imprinted ncRNA regulates gene expression remains largely unknown, it is general accepted that imprinted ncRNAs binds to chromatin modifying complexes, such as PRC2, TRX, and G9a, and generates specific silencing of genomic loci both in cis and trans. Imprinting is associated with many human diseases or syndromes (e.g. Prader-Willi, Angelman, Beckwith-Wiedemann, Retts, and Silver-Russell syndromes) and various cancers (e.g. breast, prostate, and colorectal cancers), but its role in leukemogenesis remain elusive. In this present study, the expression of a panel of 24 human imprinted ncRNA genes (AMPD3, C15orf2, COPG2, CPA4, GABRB3, H19, IGF2, IMPACT, INPP5F, L3MBTL, NR3251, NR3252, PEG3-AS, PPP1R9A, PRIM2, RASGRF1, RTL1, SFMBT2, SLC22A3, SNURF, TCEB3C, TSPAN32, ZNF215, ZNF264) and a panel of 66 human histone modifying enzymes (HME) genes was investigated in 68 newly-diagnosed acute myeloid leukemia patients with chromosome normal (AML-CN), 115 AML patients with chromosome abnormal (AML-CA), and 85 healthy individuals using real-time quantitative RT-PCR. Altered expression of 9 imprinted ncRNA genes (C15orf2, COPG2, H19, IGF2, IMPACT, PEG3-AS, PRIM2, SLC22A3, ZNF215) and 16 HME genes were observed. In AML-CN, patients’ survival days are correlated with the expression levels of H19 (p < 0.01), IMPACT (p < 0.05), DNMT3L (p < 0.05) and AURORA (p < 0.01). In AML-CA, patients’ survival days are correlated with the expression levels of PGE3-AS (p < 0.01), PRIM2 (p < 0.01), SLC22A3 (p < 0.05), and ZNF215 (p < 0.01). Multiple linear regression analysis further revealed the expression level of H19 and ZNF215 can be used as predictors for 2-year survival for AML-CN patients (p = 0.002) and AML-CA patients (p = 0.040), respectively. Cox proportional hazard model was used to analyze the hazard ratio (HR) for H19 (HR=0.868, 95.0% Confident Interval: 0.797-0.945, p = 0.001) and ZNF215 (HR=0.904, 95.0% Confident Interval: 0.821-0.995, p =0.040). In addition to survival, analysis has also been performed to correlate patients’ clinical parameters and expression levels of these altered genes and to correlate the expression levels between imprinted ncRNA genes and HME genes (results will be presented at the meeting). From our preliminary results, it is reasonable to hypothesize that loss imprinting of imprinted ncRNA is critical for the leukemogenesis of AML and under CN or CA conditions different ncRNAs are activated and affect different imprinted gene expression and thus leading to different clinical outcomes. Based on our findings, we will further perform in vitro functional analysis to elucidate the functions and mechanisms of these imprinted ncRNAs in AML tumorigenesis. Updated results of these analyzes will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 4613 Circadian rhythms regulate various functions of human body and disruption of circadian rhythm has been associated with cancer development and tumor progression. Circadian clock genes use transcriptional-translational feedback loops to control circadian rhythms. Many transcriptional regulators are histone acetyltransferases (HAT) or histone deacetylases (HDAC). As clock function and integration of inputs rely on transcriptional regulation, it is possible that chromatin is remodeled during circadian cycles and in response to signals that regulate the clock. SIRT1 (sirtuin 1) is a HDAC that has recently been identified as a crucial modulator of the circadian clock machinery. To date, at least 7 SIRT genes (SIRT1–7) have been identified. In our previous report we have demonstrated the daily expression patterns of PER1, PER2, PER3, CRY1, CRY2, and CKIe in peripheral blood (PB) of healthy individuals were abolished in chronic myeloid leukemia (CML) patients and partial recoveries of daily patterns were observed in CML patients with complete cytogenetic response (CCyR) and major molecular response (MMR) post-imatinib treatment [J Biol Rhythms 2011]. In this study we further investigated the expression profiles of the 7 SIRT genes (SIRT1–7) in PB total leukocytes from 49 CML and 22 healthy volunteers. Collection of PB was carried out at four time points: 2000 h, 0200 h, 0800 h, and 1400 h, respectively. In PB total leukocytes of healthy individuals, the daily pattern of SIRT1 (p < 0.01) and SIRT5 (p < 0.05) expression level peaked at 0200 h, and SIRT2 (p < 0.01) peaked at 0800 h. Daily pattern expression of these 3 genes was abolished in newly diagnosed pre-imatinib mesylate treated and blast crisis-phase CML patients. Partial daily patterns of gene expression recoveries were observed in CML patients with CCyR and MMR. In some serial monitored individual patients, the recoveries of oscillations of SIRT1, 2, and 5 genes expression accompanied with the disappearance of BCR-ABL transcripts were also noted. The expression of SIRT3, 6, and 7 did not show a time-dependent variation among the healthy and CML patients. SIRT4 expression was undetectable both in the healthy and CML patients. Updated in vitro study results of the regulation of SIRT1, 2, and 5 genes on circadian clock genes expression will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 3456 Studies in large-scale genome sequencing have shown that only 2% of the mammalian genome encodes mRNAs, but the most part is transcribed as long and short non-coding RNAs (ncRNAs). The ncRNAs with gene regulatory functions are starting to be seen as a common feature of mammalian gene regulation. Genomic imprinting is a form of epigenetic regulation and imprinted genes are silenced in a parental-specific manner. Imprinted genes tend to occur in clusters and ncRNAs have been found at all well-characterized imprinted clusters. Although the exact mechanism how imprinted ncRNA regulates gene expression remains largely unknown, it is general accepted that imprinted ncRNAs binds to chromatin modifying complexes, such as PRC2, TRX, and G9a, and generates specific silencing of genomic loci both in cis and trans. Imprinting is associated with many human diseases or syndromes (e.g. Prader-Willi, Angelman, Beckwith-Wiedemann, Retts, and Silver-Russell syndromes) and various cancers (e.g. breast, prostate, and colorectal cancers), but its role in leukemogenesis remain elusive. In this present study, a panel of 24 human imprinted ncRNAs genes, including ampd3, cpa4, snuf, rasgrf1, slc22a3, lgf2, treb3c, gabrb3, c15orf2, sfmbt2, rtl1, copg2, h19, l3mbtl, ppp1r9a, tspan32, lnpp5f, impact, nr3251, nr3252, znf215, prim2, peg3as and znf264, has been mined using Bioinformatics approach. We investigated the expression of these imprinted ncRNA genes using real-time quantitative RT-PCR in 67 newly-diagnosed acute myeloid leukemia patients with normal karyotypes (AML-NK), 22 AML patients with abnormal karyotypes (AML-AK), and 39 healthy individuals. In AML-NK patients, the expression of lgf2, h19, slc22a3, copg2, and impact were significantly upregulated than in healthy individuals (p < 0.0001). In AML-AK patients, besides lgf2, h19 and impact genes, ampd3 and gabrb3 were also significantly upregulated than in healthy individuals (p < 0.0001). Expression of igf2 was almost undetectable in healthy individuals but drastically increased in all AML patients. Both lgf2 and h19 were significantly increased in both AML-NK and AML-AK patients. From our preliminary results, it is reasonable to hypothesize that loss imprinting of lgf2/h19 is critical for the leukemogenesis of AML and under NK or AK conditions different additional ncRNAs are activated and affect different imprinted gene expression and thus leading to different clinical outcomes. Based on our findings, we will further perform methylation analysis of promoter CpG sites in AML patients to investigate if hypomethylation is responsible for the upregulation of these imprinted ncRNAs. We will also carry out in vitro functional analysis to elucidate the functions and mechanisms of these imprinted ncRNAs in AML tumorigenesis. Updated results of these analysis will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 3633 Circadian rhythm is present in human and all eukaryotes with a 24-hour cycle. Circadian genes use transcriptional-translational feedback loops to control circadian rhythms. Therefore, the marked characteristic of circadian systems is the strong daily cycling of clock gene mRNA, clock protein, and clock-controlled gene RNA and protein. Recent studies have demonstrated that expression of some of the circadian genes display daily oscillation in peripheral tissues including liver, eye, lung, heart, spleen, kidney, peripheral blood and bone marrow. Circadian rhythms regulate various functions of human body and disruption of circadian rhythm has been associated with cancer development and tumor progression. In this study the expression profiles of the 9 circadian genes (hPER1, hPER2, hPER3, hCRY1, hCRY2, hCLOCK, hBMAL1, hCK1e and hTIM) in peripheral blood (PB) total leukocytes from 95 chronic myeloid leukemia (CML) and 54 healthy volunteers were investigated. For comparison of circadian gene expression between PB mononuclear cells (PBMCs) and polymorphonuclear cells (PMNLs), another group of 10 healthy volunteers were also investigated. Collection of PB was carried out at four time points: 20:00, 02:00, 08:00, and 14:00, respectively. In healthy individuals, the daily oscillation was shown for hPER1, hPER2, hPER3, and hCRY2 with peak expression levels at 8:00AM, and the expression of the nine genes displayed similar profile both in PBMCs and in PMNs. In contrast, oscillations of these four genes were abolished in newly diagnosed pre-imatinib mesylate treated and blast crisis-phase CML patients and partial recoveries of oscillations were observed in CML patients with complete cytogenetic response and major molecular response. In some serial monitored individual patients, the recoveries of oscillations of circadian gene expression accompanied with the disappearance of BCR-ABL transcripts were also noted. Expression of hCRY1, hCLOCK, hBMAL1, hCK1e and hTIM did not oscillate both in healthy individuals and CML patients. Updated results on more healthy volunteers and serial monitored CML patients will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

We analyzed 23 patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) showing der(1;7)(q10;p10) (hereafter der(1;7)), to identify the exact predictive factor of this cytogenetic change. Eight (34.8%) patients, including 6 MDS and 2 AML patients, had a prior history of genotoxic exposure, especially radiation and/or antimetabolites. Patients with der(1;7) consisted of three groups; one-third of patients had a prior history of genotoxic agents, one-third had additional cytogenetic changes at the time of MDS/AML diagnosis without prior exposure history, and the remaining one-third had neither prior exposure history nor additional cytogenetic changes. The current study demonstrated that the poor outcome of MDS/AML with der(1;7) is due to the high frequency of associated risk factors, i.e., prior history of genotoxic exposure, the presence of additional cytogenetic changes, or both. Identification of prognostic disadvantage might be required for appropriate strategy in managing MDS/AML patients with rare der(1;7) abnormality.

Circadian rhythm is present in all eukaryotic and some prokaryotic life forms. This time-keeping system is organized in a hierarchical fashion and composed of a self-sustained central pacemaker in the suprachiasmatic nuclei of the anterior hypothalamus and peripheral oscillators in most body cells. A recent study demonstrated that mice deficient in the clock gene mPer2 are cancer prone and display a deregulated temporal expression genes involved in cell cycle regulation, such as c-Myc, Cyclin D1, Cyclin A, and Mdm-2. These mice display salivary gland hyperplasia, and a large portion of them develops lymphomas. The results revealed that Per2 is an essential circadian gene and it is associated with proliferation control in mammals. In a more recent study, it was demonstrated that an age-dependent decay of the circadian clock both at the behavioral and the molecular levels was observed in mPer1−/−mCry2−/− double-mutant mice. To gain further insights into the roles of circadian genes in chronic myeloid leukemia (CML), we analyzed peripheral blood from 21 healthy individuals and 35 CML patients (18 in blastic crisis and 17 in chronic phase) by real-time quantitative RT-PCR for the expression of circadian genes PER1, PER2, and PER3. In blastic crisis cases, the expression levels of all three PER genes were significantly impaired than in healthy individuals (PER1, 1:5.97, p < 0.005; PER2, 1:13.51, p < 0.0001; PER3, 1:2181.33; p < 0.0001); whereas, in chronic phase, only the expression levels of PER2 and PER3 were significantly impaired (PER1, 1:1.23, p > 0.5; PER2, 1:2.47, p < 0.05; PER3, 1:14.22; p < 0.0001). Mutational analysis of the whole gene and methylation analysis of CpGs sites at the promoter area were further performed to investigate the possible mechanisms. No mutation was found within the coding regions of the three PER genes in all CML cases. Methylation analysis using methylation-specific PCR and direct sequencing showed no methylation in the promoter areas of both PER1 and PER2 genes. In contrast, most of the CpG sites were methylated in the promoter area of PER3 gene in CML cases and none of these CpG sites were methylated in healthy individual cases. In addition, the methylated CpG frequencies of PER3 gene differed in patients at blastic crisis and at chronic phase (CpG, 8.24 ± 0.73 vs. 4.48 ± 0.48, p < 0.0001; T/CpG, 10.47 ± 0.67 vs. 14.67 ± 0.46, p < 0.0001; TG, 1.24 ± 0.32 vs. 0.81 ± 0.16, p > 0.05). Demethylation by treatment of hypermethylated K562 cells with 5′-aza-2′-deoxycytidine resulted in partial reactivation of PER3 expression. Our results suggest that the downregulated PER3 expression in CML was due to the inactivation of PER3 gene by methylation and the differential expression was correlated to the ratio of methylated CpG sites at the promoter region. We hope to explore the role of circadian genes in the tumorigenesis of leukemia and to establish circadian genes as novel and useful markers for diagnosis and references for therapies in leukemia.