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The RNA splicing factor SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL). Co-expression of mutant Sf3b1 and Atm deletionin murine B cellsresults in CLL development, in which CLL cells exhibit RNA splicing dysregulation, increased DNA damage and recurrent chromosomal amplification. These findings suggest a strong link between splicing factormutation, chromosomal instability (CIN), and oncogenesis, although the mechanism has yet to be elucidated. CIN in cells with defective RNA processing is triggered, at least in part, by excessive R-loop (RNA:DNA hybrid with displaced single strand DNA). Using dotblot assay, we observed R-loop augmentation in SF3B1 mutant (MT) compared to wild-type (WT) cells in both human cell lines and murine CLL cells. Along with R-loop accumulation, SF3B1 MT increased DNA double strand breaks (DSB) (measured by comet assay) and mis-segregated chromosomes during mitosis (quantified by immunofluorescence using anti-phospho-histone H3 and anti-αtubulin antibodies). Overexpression of R-loop resolving enzyme RNaseH1 in SF3B1 MT cell lines reduced DSB (t-test p-value To elucidate the mechanisms underlying SF3B1 MT-associated CIN, we used DNA:RNA hybrid immunoprecipitation (DRIP) coupled with sequencing to map R-loop in SF3B1 MT and WT Nalm6 cells. Although SF3B1 MT caused minimal changes in overall R-loop distribution, it led to increased R-loop formation at centromeric regions. Proper centromeric R-loop (cen-R-loop) formation is essential for accurate chromosome segregation. Those R-loop are coated by phosphorylated replication protein A (p-RPA). p-RPA and centromere immunofluorescence (IF) co-staining revealed increased cen-R-loop in SF3B1 MT cells. Intriguingly, overexpression of RNaseH1 reduced both levels of centromeric p-RPA and percentage of cells with defective mitosis, suggesting SF3B1 MT associated cen-R-loop impacts CIN. To further discern the molecular underpinnings of R-loop mediated CIN, we investigated the effect of their accumulation on spindle geometry in metaphase cells. By analyzing the total body of chromosomes two-dimensional area, we observed one fold greater magnitude of chromosome oscillations in SF3B1 Nalm6 MT cells. Accordingly, compared to WT cells, SF3B1 MT cells displayed longer and wider spindles to accommodate an aneuploid chromosomal content. Removal of R-loops rescued mitotic spindle architecture and CIN in SF3B1 MT cells, highlighting a direct contribution of R-loop to kinetochore-microtubule organization and stability. As there is no evidence of strong binding of SF3B1 to centromeres, we tested the possibility that SF3B1 MT contributes to cen-R-loop formation through alternative splice variants. Through overlapping conserved SF3B1 MT-associated splice variants across isogenic cell lines and murine model with available R-loop interactome data, we identified SERBP1, which encodes an RNA-binding protein, as a target candidate . SF3B1 MT induces loss-of-function in SERBP1 through alternative splicing. Knockdown of SERBP1 triggered R-loop accumulation, increased centromeric p-RPA coating, and aberrant mitosis, which recapitulated the SF3B1 MT phenotype. Importantly, overexpression of SERBP1 WT alleviated both DSB and R-loop accumulation in SF3B1 MT cells. These observations strongly suggest that SF3B1 MT augments R-loop formation through dysregulated splicing of SERBP1 and possibly other genes involved in R-loop metabolism. We also investigated the possibility that the impact of RNA splicing defect on R-loop biology is a generalizable phenomenon. Examing a panel of leukemia-associated splicing factors (U2AF1, SRSF2, ZRSR2, RNU1), we found that these mutations all display R-loop dependent mitotic stress. Thus, mitotic stress caused by aberrant R-loop formation could be a convergent mechanism in splicing factor mutated leukemia cells. Altogether, we demonstrated that SF3B1 mutation promotes CIN by destabilizing mitotic spindles through aberrant R-loop accumulation at the centromere, via dysregulated splicing in genes such as SERBP1. Our study highlights an unrecognized role of cen-R-loop as a critical link between RNA splicing dysregulation and CIN, providing an opportunity for therapeutic targeting of R-loop in splicing factor mutant leukemias. Disclosures Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Although large-scale sequencing studies have elucidated key mutations in chronic lymphocytic leukemia (CLL), the molecular pathways underlying CLL transition to aggressive lymphoma (Richter's transformation, RT) are not completely understood. Co-expression of the two most common human CLL genomic alterations, 13q deletion, and SF3B1 mutation, in murine B cells results in indolent CLL. Seventy percent of RT cases involve MYC network aberrations, and MGA (Max-gene-associated), a functional MYC suppressor, is recurrently mutated/deleted in ~10% of RT cases. Given the function of MGA in MYC dysregulation, we sought to determine if loss-of-function MGA mutations accelerate CLL transformation. To determine the function of Mga deletion in CLL-RT, we in-vitro edited the genome of LSK (Lin - Sca-1 + Kit +) cells derived from the conditional CD19Cre/Cas9(GFP)/Mdr/Sf3b1-expressing mice (donor mice:CD45.2) to introduce Mga deletion in B cells with Mdr and the Sf3b1K700E mutations. Mga and control sgRNAs were lentivirally transduced in LSK cells and then transplanted into sub-lethally irradiated recipient mice (N=15, per group; C57BL/6: CD45.1). CLL onset (B220 +CD5 + CLL-like cells) was monitored bimonthly by flow-cytometry of peripheral blood, between ages 6 and 24 months. Three of 15 mice with Mga deletion had a clonal expansion of B220 +CD5 + cells with leukemic cells infiltration in the spleen and bone marrow by 21-months of age. However, no RT signs were observed in these mice. Transplantation of the CLL-like splenic cells into immunocompromised NSG or immunocompetent sub-lethally irradiated CD45.1 mice, led to rapid expansion of B220 + cells along with CD5 loss at 3 weeks post-engraftment and leukemic cells infiltrated various lymphoid tissues. H&E staining of splenic sections revealed the presence of CLL-like cells with a morphology resembling leukemia in the primary engrafted mice; however, these cells were larger and resembled aggressive lymphoma (RT) upon secondary engraftment. CLL and RT features were further confirmed by IHC staining of PAX5, CD5, Ki67, and MYC, suggesting the establishment of a murine CLL-to-RT transition murine model. MYC was reported to contribute to cell growth by promoting mitochondrial dysfunction. To characterize mitochondrial changes in our RT model, oxidative phosphorylation (OXPHOS) was measured by Seahorse, and electron microscopy was performed on RT and no disease splenic B cells. Murine RT cells exhibited increased basal respiration and mitochondrial mass coupled with elevated mitochondria number and structural changes. To pinpoint the underlying transcriptional program, we performed RNA seq using splenic B cells derived from control, CLL, and RT mice. Through differential gene expression analysis, we identified >200 genes (mainly hallmark MYC targets and OXPHOS associated pathways) associated with CLL and RT. Of the 75 upregulated genes shared between CLL and RT cells, we focused on Nme1 (Nucleoside diphosphate kinase), an MGA transcriptional target reported in human lung adenocarcinoma. We confirmed Nme1 upregulation at both RNA and protein levels in murine and human RT samples by RT-PCR, immunoblot, and IHC, implicating a role of NME1 during CLL to RT transition. To determine if the MGA/MYC/NME1 axis can be recapitulated in human cells, we generated combined genetic lesions in two human B-cell lines (harboring SF3B1K700E Nalm6E [E] or control Nalm6K [K]) by Crispr-Cas9 mediated deletion of MGA and MDR. In both MGA and MDR-MGA KO(Knockout) cells, NME1 RNA and protein were upregulated. MGA KO E cells displayed a mixture of large and small-sized cells with increased cell growth whereas K cells had no morphology change. Moreover, large E cells displayed increased mitochondria mass, basal and maximal OXPHOS, broken cristae, fully recapitulating the mitochondrial dysfunction observed in murine RT cells. Harnessing these cell lines, we discovered that NME1 KO diminished cell growth and OXPHOS in E/K cells, which can be rescued by NME1 overexpression, highlighting an essential but unrecognized role of NME1 in driving the CLL-RT transition. With our established murine CLL-to-RT model, we demonstrated an RT-associated mitochondrial phenotype and revealed a novel role of the MGA/MYC/NME1 axis in driving the CLL-to-RT transition through mitochondrial regulation. This study opens a new therapeutic avenue for RT patients by targeting the MGA/MYC/NME1 axis. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Speakers Bureau; Oncternal: Research Funding; TG Therapeutics: Research Funding. Danilov: Abbvie: Consultancy, Honoraria; Takeda Oncology: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; TG Therapeutics: Consultancy, Research Funding; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria; Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding.

RNA splicing factor SF3B1 is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL), but expression of this mutation alone in murine B cells does not result in CLL. This gene mutation is often subclonal and associated with poor survival. How this mutation impacts CLL progression remains elusive. Since SF3B1 mutation frequently co-occurs with chromosome 13q deletion (del(13q)), and mice with deletion of the Minimal Deleted Region (MDR) of del(13q) develop indolent CLL, we therefore asked whether co-expression of Sf3b1 mutation can accelerate the onset of CLL in this murine model. If so, how does Sf3b1 mutation mechanistically contribute to CLL. To this end, we first crossed mice carrying conditional knock in allele Sf3b1-K700E and mice with conditional knockout of MDR. We then bred the offspring with CD19-Cre mice to generate cohorts of mice which have B cell-specific homozygous deletion of MDR with (DM) or without (MDR-MT) heterozygous Sf3b1-K700E. We monitored the onset of CLL by tracking of circulating B220+CD5+ CLL-like cells from peripheral blood with flow cytometry, starting at the age of 6-months and ending by 24-months. We detected CLL-like disease in 24% (6 of 25) of DM and 7.4% (2 of 27) of MDR-MT mice with disease presence in the spleen, bone marrow and lymph node, confirmed by flow cytometry and immunohistochemistry. The increased frequency of CLL in DM mice indicated that Sf3b1-K700E could accelerate CLL (Pearson Chi-Square 2-sided, p=0.098). To elucidate how Sf3b1 mutation contributes to increased CLL penetrance, we performed integrated RNA sequencing (RNA-seq) and TMT proteomics analysis with splenic B cells derived from DM mice with and without CLL. We found that genes involved in MYC, cell cycle checkpoints and mTORC1 pathways are upregulated and enriched at both the RNA and protein levels when we compared DM-CLL cells to their DM B cell counterparts, indicating these cellular processes are involved in the onset of CLL. To further define the role of Sf3b1-K700E mediated alternative splicing in the activation of these pathways, we first identified candidate splicing isoforms (nfatc1, braf, depdc5, tsc2) through computational analysis of RNA-seq data and then validated the isoforms in an independent cohort of samples (n=3,). Functional annotation of how exactly these isoforms impact CLL is ongoing. Importantly, we also observed gene upregulation of mTORC1 pathway in human CLL cells with SF3B1 mutation and del(13q) when compared with normal B cells. We next asked whether DM CLL cells are sensitive to inhibition of mTORC1 pathway and RNA splicing inhibition in vitro. We exposed DM B and CLL cells to either Temsirolimus (Tem, mTORC1 inhibitor), or H3B8800 (H3B, SF3B1 inhibitor) alone or in combination for 24 hours and then measured the cell viability with CellTiter-Glo assay. When compared to DMSO control, both Tem and H3B single treatments significantly inhibited the survival of DM CLL cells, but not DM B cells (all groups vs control, unpaired t test, p Our study demonstrates that expression of Sf3b1-K700E could accelerate the development of CLL based on MDR deleted murine model through alternative RNA splicing and mTORC1 activation. This finding supports the use of an mTORC1 inhibitor together with RNA splicing inhibitor in the subset of CLL patients with both SF3B1 mutation and del(13q). Disclosures Kipps: Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding. Neuberg:Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

Background and purpose: Eltrombopag (ELT) was effective in the treatment of relapse/refractory aplastic anemia (AA) patients. Response and adverse drug reaction (ADR) differed greatly among individuals even at the same dosage of ELT. Methods: Patients diagnosed with non-severe aplastic anemia (NSAA) between January 2018 to January 2019 in Peking Union Medical Colleague Hospital who were refractory to immunosuppressive therapy were treated with ELT and followed up for at least 6 months. Plasma concentrations of ELT were detected by the high-performance liquid chromatography-mass spectrometry after at least two months of ELT treatment and staying for that dosage for at least two weeks. Dose-concentration, concentration-response and concentration- ADR relation were evaluated. Results: Among the 72 patients treated with ELT during that period, 44 patients with complete data were enrolled. 6 (13.6%) were males and 38 were females (86.4%) with a median age of 54 years (IQR: 38.5-63). By the time of plasma concentration detected, the median dosage of ELT was 75(IQR 50-100) mg/d and median time for total ELT exposure was 3 (IQR 2.0-6.0) months, and 37 (70.5%) patients responded to ELT. The median concentration of ELT was 10.4μg/ml (IQR 3.7-24.4μg/ml). The concentration of ELT was positively correlated with the daily dose of ELT (r = 0.68, p < 0.001). Multivariate logistic regression analysis showed that the risk of inefficacy of ELT with a concentration between 11.2-15.2 μg/ml was 0.028-fold (95% CI: 0.001-0.864; P=0.041) to those with a concentration between 3.2-7.2μg/ml. The cutoff value of concentration for ELT efficacy was 12.50μg/ml by receiver operation characteristic curve. A higher risk of ADR was related to a longer total exposure to ELT (P=0.012). Although not significant, odd ratio (OR) increased with ELT concentration, suggesting a possible elevated risk of ADR correlated with blood concentration. Conclusions: ELT is effective for the treatment of NSAA with accepTable side effects. Plasma concentration of ELT correlated with the dose and the effects of ELT. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Eltrombopag (ELT) has been proven to induce trilinear hematopoietic responses in relapse/refractory AA patients and has been approval as a monotherapy in relapsed/refractory SAA in the USA and Europe. However, the use of ELT is only approved for idiopathic thrombocytopenic purpura (ITP) in China by the National Medical Products Administration (NMPA), for AA it is still "off-label".

One of the most common secondary resistance mechanisms to ibrutinib (IBR, a first-generation, irreversible Bruton's tyrosine kinase [BTK] inhibitor) in CLL is the development of mutations in BTK involving Cys481, leading to impaired drug binding (Woyach et al, NEJM 2014; Furman et al, NEJM 2014). The mechanisms of resistance to second generation BTK inhibitors are currently unknown. We aimed to assess the spectrum of acquired BTK mutations in patients with CLL progression on zanubrutinib (ZANU), a second-generation, irreversible inhibitor of BTK. We identified 38 CLL patients, treated with ZANU on clinical trials (NCT02343120, NCT02569476, NCT03336333, NCT02795182) at three centres, for whom serial samples were available. Four of 38 patients (10.5%) had CLL progression on ZANU (time to progression 5, 26, 29 and 48 months) and underwent amplicon next generation sequencing (NGS) of BTK (exon 11, 15, 16) and PLCG2 (exon 16, 19-20, 24, 27-28). Remarkably, we detected a BTK kinase domain mutation, BTK Leu528Trp (NM_000061.2:c.1583T>G), in all four patients progressing on ZANU. In addition, all four patients had detectable Cys481 mutations at lower variant allele frequency (VAF) than the BTK Leu528Trp (median BTK Leu528Trp 34.9% vs BTK Cys481 9.1%). Analysis of sequence reads from amplicon NGS and RNA-sequencing data demonstrated that BTK Leu528Trp and BTK Cys481 mutations were present on different alleles. Assessment of the BTK Leu528Trp and BTK Cys481 mutations with high sensitivity droplet digital PCR (ddPCR) confirmed the absence of both mutations prior to ZANU exposure in all patients (sensitivity 0.1% VAF). Longitudinal analysis of the four patients with the BTK Leu528Trp mutation demonstrated the appearance of the Leu528Trp coincident with rising measurable disease and subsequent clinical CLL progression. We then went on to test patients on ZANU without disease progression but with persistent measurable disease (n=34) by ddPCR and detected three further patients harbouring low level BTK Leu528Trp mutations (VAF The BTK Leu528Trp mutation has been described only once previously in a patient in the context of IBR resistance (who transformed with Richter's syndrome) where it co-occurred with Cys481 mutations (Maddocks et al, JAMA Oncol 2015). As the prevalence of BTK Leu528Trp among progressive disease samples in our cohort exceeds all prior reports in IBR-treated patients, we sought to further understand the specificity of BTK Leu528Trp for ZANU progression. Targeted sequencing in a cohort of 49 patients progressing on IBR from the European Research Initiative on CLL (ERIC) did not detect the BTK Leu528Trp in any patients (sensitivity 1% VAF). We went on to perform biochemical and cellular studies on the BTK Leu528Trp mutation. Assessment of enzymatic activity of BTKLeu528Trp demonstrated a significant loss of activity compared to both BTKWT and BTKCys481Ser. This was further confirmed by assessing BTK autophosphorylation in HEK293 cells. Autophosphorylation at BTK Tyr223 was markedly reduced in HEK293 cells stably expressing BTKLeu528Trp compared to both BTKCys481Ser and BTKWT. In addition, a crystal structure of apo-BTKLeu528Trp was solved to understand effects of BTKLeu528Trp on ZANU binding to BTK. The alignment of the crystal structure of apo-BTKLeu528Trp with that of BTKWT-ZANU or the modeled structure of BTK-ATP suggested potential steric clashes between BTKLeu528Trp and ZANU (Figure 1A), as well as BTKLeu528Trp and ATP (Figure 1B). In conclusion, we have described the novel enrichment of BTK Leu528Trp mutations occurring in patients with CLL progressing on ZANU and both structural and experimental data consistent with this mutation resulting in interference with both ATP and ZANU binding to BTK. These findings emphasize the potential for agent-specific resistance mutations with second generation BTK inhibitors and the need to include these mutations in diagnostic screening for BTK resistance in the clinic. SH/CPST co-first authors, CT/PB co-senior authors Disclosures Handunnetti: Abbvie: Other: Travel Grant; Gilead: Honoraria. Zhou:Beigene: Employment. Sun:Beigene: Employment. Xing:Beigene: Employment. Zhang:Beigene: Employment. Guo:Beigene: Employment. Sutton:Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Ghia:Dynamo: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy; Novartis: Research Funding; Juno/Celgene: Consultancy, Honoraria. Scarfo:AstraZeneca: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Seymour:Takeda: Consultancy; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; Janssen: Research Funding; BeiGene: Research Funding. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Liu:Beigene: Employment. Cheah:Roche, Janssen, MSD, Gilead, Loxo Oncology, Acerta, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Abbvie: Research Funding; Roche: Other: Travel expenses. Tam:Janssen: Honoraria, Research Funding; BeiGene: Honoraria; Pharmacyclics LLC, an AbbVie company: Honoraria; Roche: Honoraria; Novartis: Honoraria; AbbVie: Honoraria, Research Funding. Blombery:Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy.

Reprogramming of somatic cells to desired cell types holds great promise in regenerative medicine. However, production of transplantable hematopoietic stem cells (HSCs) in vitro by defined factors has not yet been achieved. Therefore, it is critical to fully understand the molecular mechanisms of HSC development in vivo. Here, we show that Fev, an ETS transcription factor, is a pivotal regulator of HSC development in vertebrates. In fev-deficient zebrafish embryos, the first definitive HSC population was compromised and fewer T cells were found in the thymus. Genetic and chemical analyses support a mechanism whereby Fev regulates HSC through direct regulation of ERK signaling. Blastula transplant assay demonstrates that Fev regulation of HSC development is cell autonomous. Experiments performed with purified cord blood show that fev is expressed and functions in primitive HSCs in humans, indicating its conserved role in higher vertebrates. Our data indicate that Fev-ERK signaling is essential for hemogenic endothelium-based HSC development.

Epigenetic drugs are currently used for the treatment of several hematologic malignancies, but their mechanism of action remains poorly understood. By using a previously described reporter cell line for epigenetic reactivation of the DAPK1 locus, we have shown that epigenetic treatment causes transcription from uncharacterized intronic transcription start sites (TSSs), thereby generating DAPK1 mRNA with novel first exons. Based on these findings, we analyzed whether inhibition of DNA-Methyltransferases (DNMTs), Histone deacetylases (HDACs), or both resulted in the genome-wide induction of non-canonical TSSs. While epigenetic treatment altered expression of known promoter sites, we observed that both HDAC- and DNMT-inhibitors predominantly induced de novo transcription from cryptic promoters encoded in long-terminal repeat (LTR) retrotransposons. These LTR-associated 'treatment induced, not-annotated TSS' (TINATs) are currently not annotated and normally silenced in almost all cell types with the exception of testicular und thymic tissue. In the majority of cases, these TINATs arose most commonly from LTR12 elements, particularly LTR12C (which apparently provides 50% of all TINATs). TINAT activation after DNMT-inhibitors (DNMTi) coincided with DNA hypomethylation and gain in H3K4me3, H3K9ac, and H3K27ac histone marks. In contrast, HDAC-inhibitors (HDACi) induced only canonical TSSs in association with histone acetylation, but TINATs via a yet unknown mechanism. Nevertheless, both inhibitors convergently induced unidirectional transcription from identical TINAT sites. Moreover, we found a consensus GATA2 binding motif which strongly distinguished LTR12Cs with TINATs from LTR12Cs without TINATs, supporting that GATA2 is likely the upstream transcription factor responsible for TINAT activation. TINATs originating from non-canonical TSSs located within introns of protein-coding genes frequently spliced into downstream exons thereby creating LTR/non-LTR fusion transcripts that harbor novel in place of canonical exon sequence at their 5' end. The resulting transcripts encode truncated or chimeric open reading frames which translated into currently uncharacterized protein isoforms with predicted abnormal functions or immunogenic potential, the last one based on their foreign sequence and capability of being presented on MHC-class I molecules. In summary, we could show that DNMTi and/or HDACi do not predominantly alter the expression of canonical genes, but induce de novo transcription of LTRs especially of the LTR12 family, resulting in numerous fusion transcripts that encode novel protein isoforms which might have the potential to influence cell proliferation or might be an elegant explanation for the priming effect of epigenetic therapy. Ongoing experiments are investigating the functional mechanisms of TINAT reactivation upon epigenetic drug treatment and future proteomic approaches combined with T-cell cytotoxicity assays will further shed light on the interaction between epigenetic and immune therapy and the role of ERV-derived antigen presentation. Disclosures Lübbert: Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding.

To elucidate the molecular mechanism of all-trans-retinoic acid (ATRA)–induced differentiation of acute promyelocytic leukemia (APL) cells, the gene expression patterns in the APL cell line NB4 before and after ATRA treatment were analyzed using complementary DNA array, suppression-subtractive hybridization, and differential-display–polymerase chain reaction. A total of 169 genes, including 8 novel ones, were modulated by ATRA. The ATRA-induced gene expression profiles were in high accord with the differentiation and proliferation status of the NB4 cells. The time courses of their modulation were interesting. Among the 100 up-regulated genes, the induction of expression occurred most frequently 12-48 hours after ATRA treatment, while 59 of 69 down-regulated genes found their expression suppressed within 8 hours. The transcriptional regulation of 8 induced and 24 repressed genes was not blocked by cycloheximide, which suggests that these genes may be direct targets of the ATRA signaling pathway. A balanced functional network seemed to emerge, and it formed the foundation of decreased cellular proliferation, maintenance of cell viability, increased protein modulation, and promotion of granulocytic maturation. Several cytosolic signaling pathways, including JAKs/STAT and MAPK, may also be implicated in the symphony of differentiation.

The ABL tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM) is effective at inducing clinical remission in early phase chronic myeloid leukemia (CML) patients, but is not curative. Early relapses and acquired drug resistance remain some issues in IM-treated patients. In particular, relapses are frequently associated with point mutations in the BCR-ABL tyrosine kinase domain (TK, > 50%). Newer TKIs, dasatinib (DA) and nilotinib, have increased potency over IM and show a broader spectrum of activity against mutant forms of BCR-ABL. However, none of these agents is able to target a critical T315I gatekeeper mutation of BCR-ABL in TKI-resistant patients. We have demonstrated that CML stem cells are genetically unstable and generate many BCR-ABL TK mutations in vitro and in vivo. They are also less responsive to TKIs and are a critical target population for TKI-resistance. Thus improved treatment approaches to specifically target CML stem cells and BCR-ABL-T315I resistant cells are clearly needed. It has recently been reported that estrogen receptor variant ERα36 is highly deregulated not only in breast cancer cells, but also in liver cancer and leukemic cells, and that targeting this specific variant with a small molecular inhibitor (Icaritin, SNG162) inhibits CML cell growth. However, the underlying molecular mechanisms of these observations are not understood. Whether this inhibitor, alone or in combination with new ABL inhibitors, can target primary CML stem/progenitor cells and T315I-resistant cells have also not been investigated. In this study, we utilized two cell line model systems: K562 cells and IM-resistant K562 cells without BCR-ABL TK mutation and human UT7 cells expressing either wild type BCR-ABL or carrying the BCR-ABL-T315I mutation. We have now demonstrated that protein expression of ERα36 is highly upregulated in both IM-resistant and BCR-ABL-T315I mutant cells as compared to control cells. Interestingly, the use of pre-clinically validated ERα36 inhibitors (SNG162 and SNG1153) alone inhibits cell growth and induces apoptosis of these cells. BCR-ABL-T315I cells are more sensitive to ERα36 inhibitors treatment, with twofold increases in Annexin V+ cells detected in BCR-ABL-T315I cells after SNG1153 treatment, compared to those detected in BCR-ABL expressing cells. These effects can be further enhanced by combination treatment with a TKI. Importantly, we have discovered that treatment of IM-resistant and BCR-ABL-T315I mutant cells with SNG162 and SNG1153 inhibitors, alone or with a TKI, significantly reduced phosphorylation of BCR-ABL on tyrosine residue 177 (Tyr177), a residue essential for BCR-ABL induced leukemogenesis through its binding to GRB2 and activation of the downstream RAS-MAPK pathway. This new observation was supported by detection of a significant reduction in phosphorylation of MEK1/2 kinase, an important component of the RAS-MAPK pathway, in these cells. Most importantly, IP-Western analysis further demonstrated that the BCR-ABL-GRB2 protein interaction was markedly interrupted in cells treated with SNG inhibitors plus a TKI, which correlates with reduced phosphorylation of Tyr177 in these cells. Moreover, colony-forming cell (CFC) assays showed that SNG inhibitors (SNG162 and SNG1153) in combination with a TKI are more effective at inhibiting growth of CD34+ treatment-naïve IM-nonresponder cells as compared to single drug treatment (46% vs. 25%). We further demonstrated that SNG162 and SNG1153 (up to 10 μM and 5 uM) are not toxic to CD34+ normal bone marrow cells. Interestingly, a combination treatment of DA plus SNG1153 dramatically reduced the level of engrafted leukemic cells in transplanted immunodeficient NSG mice and prolonged the survival of these mice compared to mice treated with DA alone. Median survival of DA treated mice is 87.5 days, while some DA and SNG1153 treated mice remain alive for more than 102 days after transplantation. ERα36 thus emerges as an attractive druggable target for combination therapies to target TKI-insensitive CML stem/progenitor cells and T315I-resistant cells. Disclosures Zhang: Shenogen Pharma Group Ltd: Employment. Ding:Shenogen Pharma Group Ltd: Employment. Lin:Shenogen Pharma Group Ltd: Employment. Meng:Shenogen Pharma Group Ltd: Employment.

Abstract 5025 The CRBN gene that encodes the cereblon protein is found on the short arm at position p26. 3 of human chromosome 3. Cereblon is a primary target of thalidomide teratogenicity and required for the anti-myeloma activity of lenalidomide and pomalidomide. CRBN depletion myeloma cells become highly resistant to both lenalidomide and pomalidomide. Baicalein, a component of Scutellaria radix from HLJDT, not only suppressed proliferation and induced apoptosis of myeloma cells by down-regulating interleukin −6(IL-6) and XIAP gene expression, but also inhibited the signaling cascades mediated by IL-6 and facilitated myeloma cell inhibition induced by dexamethasone. In clinic, we found that treatment of thlidomide- or lenalidomide-resistant myeloma patients by applying Huang-Lian-Jie-Du-Tang (HLJDT) can induce hematological remission. The precise molecular mechanism of HLJDT exerts its anti-tumor effects remains unclear. Here, by RT-PCR, we demonstrated that treatment of U266 cells and primary myeloma cells with 20μM baicalein can induce CRBN mRNA expression in time-dependent manner. As lenalidomide and thlidomide are effective drugs for maintenance therapy with the advantage of oral administration. It was particularly active in patients with higher cereblon expression. Thus, the combination of HLJDT with thlidomide or lenalidomide may be a novel strategy of maintenance therapy for myeloma patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4361 Occupying the ATP binding pocket represents a common strategy for tyrosine kinase inhibition, exemplified by imatinib mesylate in BCR-ABL positive leukemia and gefitinib in EGFR mutated lung cancer. Here we report that the natural compound triptolide and proteasome inhibitor Bortezomib induce C-KIT turnover in cancer cells. Bortezomib triggers a clathrin-mediated endocytosis and lysosomal degradation of KIT in t(8;21) leukemia. While phosphorylation of heat shock protein 90 (Hsp90) by KIT is required for Hsp90-caused suppression of apoptosis, Bortezomib causes dephosphorylation of Hsp90 and lease of Apaf-1/cytochrome C, leading to reactivation of caspase-3 which cleaves the t(8;21)-generated AML1-ETO/AML1-ETO9a oncoproteins with production of cleavage fragments. The cleavage fragments antagonize the parental proteins in vitro and in vivo. Intriguingly, Bortezomib shows potent anti-leukemia efficacy in two murine models. These data indicate that degradation of C-KIT/dephosphorylation of Hsp90 represents a novel strategy for kinase inhibition, and drugs inhibiting both transcription factor and kinase could be a powerful therapeutic regimen for human leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 1007 Poster Board I-29 In our previous study, we showed that the hypoxia or hypoxia-mimetic agents induces differentiation of myeloid leukemic cells. During this event, hypoxia-inducible factor-1a (HIF-1a) exerts a critical role in its transcriptional activity-independent manner. MicroRNAs (miRNAs) regulate the expression of 30% of the transcripts of human genome on a posttranscriptional level. Recent work on miRNA has shed light on the possible involvement of miRNA genes in biological processes such as cell proliferation, differentiation and hematopoiesis. To reveal the miRNA expression profile and related roles in HIF-1a-induced differentiation of myeloid leukemic cells, miRNA expression profiling was analyzed by microarray in HIF-1a-expressing U937T cells (U937Tclone) and HIF-1a-absent cells (U937Tempty) in 0 day, 2 days and 4 days after tetracycline removal. The differentially expressed miRNAs, as verified by quantitative real-time RT-PCR and Northern blot, were subjected to gene ontology (GO) analysis and pathway analysis. The upregulated and downregulated miRNAs in U937Tclone cells in three time points after tetracycline removal were 11 miRNAs and 7 miRNAs, respectively. The differential expression of miR-26a, -107, -19b, -20a, -17, -92a, -92b and -106a was validated. On the basis of bioinformatic analysis, we focused on the changed components of miRNA 17-92 cluster and miRNA 106a-92 cluster, mostly considered as oncomirs, which have the similar downregulated expression profile. The results showed that miR-17 was significantly depressed by 40% and 70% in 2 and 4 days after tetracycline removal in U937Tclone. And miR-20a was significantly depressed by 60% and 80%, respectively. High-enrichment GOs containing differentiation-related targeted genes and miRNA-gene networks revealed STAT3 as one of the highest degree of target genes, which was targeted by the three changed components of miR-17 family (miR-17, -20a and -106a). STAT3 protein was validated to be upregulated in U937Tclone after tetracycline removal by Western blot, and no fluctuation in STAT3 mRNA levels was observed, which may uncover the critical role of miR-17 family in the cell differentiation induced by HIF-1a. In summary, significantly different miRNA profiles were seen in U937Tclone cells as compared to U937Temptycells. The downregulation of the components of miRNA 17-92 and 106a-92 cluster may contribute to HIF-1a-induced myeloid leukemic cell differentiation by targeting STAT3 or other signaling pathways. Further functional study of these miRNAs will be carried out by overexpression and knockdown experiments. These data would shed new insights for understanding mechanisms underlying leukemogenesis and designing the new therapeutic strategy for differentiation induction of acute myelogenous leukemia. Disclosures: No relevant conflicts of interest to declare.

Bruton’s tyrosine kinase (BTK), a member of the Tec family of nonreceptor protein tyrosine kinases, is critical for early B cell development and mature B cell activation and survival, and is downregulated in plasma cells. Mutation of BTK leads to human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). Here we investigate BTK function in multiple myeloma (MM) cells. Although rare mutations were found in the promoter and coding sequence of the BTK gene, elevated BTK at mRNA level was detected in MM cell lines and CD138 positive primary cells from 3 out of 8 cases of patients with MM. At protein level, BTK was upregulated in some of MM as well as myeloid leukemia cell lines. Specific inhibitor of BTK also suppressed proliferation of MM cells. These data suggest a role for BTK to play in MM pathogenesis.