MDS-L Cells Research Articles

Enhanced ALOX12 Gene Expression Predicts Therapeutic Susceptibility to 5-Azacytidine in Patients with Myelodysplastic Syndromes.

5-azacytidine (AZA), a representative DNA-demethylating drug, has been widely used to treat myelodysplastic syndromes (MDS). However, it remains unclear whether AZA's DNA demethylation of any specific gene is correlated with clinical responses to AZA. In this study, we investigated genes that could contribute to the development of evidence-based epigenetic therapeutics with AZA. A DNA microarray identified that AZA specifically upregulated the expression of 438 genes in AZA-sensitive MDS-L cells but not in AZA-resistant counterpart MDS-L/CDA cells. Of these 438 genes, the ALOX12 gene was hypermethylated in MDS-L cells but not in MDS-L/CDA cells. In addition, we further found that (1) the ALOX12 gene was hypermethylated in patients with MDS compared to healthy controls; (2) MDS classes with excess blasts showed a relatively lower expression of ALOX12 than other classes; (3) a lower expression of ALOX12 correlated with higher bone marrow blasts and a shorter survival in patients with MDS; and (4) an increased ALOX12 expression after AZA treatment was associated with a favorable response to AZA treatment. Taking these factors together, an enhanced expression of the ALOX12 gene may predict favorable therapeutic responses to AZA therapy in MDS.

Distinct Role of STAT3 and STAT5 Factors in MDS and AML

Introduction: Myelodysplastic Syndrome (MDS) refers to a heterogeneous group of clonal hematologic disorders characterized by hematopoietic dysfunctions and impaired differentiation in the hematopoietic stem and progenitor cells compartment. MDS is commonly referred to as a pre-leukemic stage due to the increased risk of progression to Acute Myeloid Leukemia (AML). AML is an aggressive hematologic malignancy characterized by the accumulation of immature myeloblasts in the bone marrow and the peripheral blood. Signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) are important regulators of several cellular processes including cell proliferation and survival. Abnormal STAT3 and STAT5 signaling has been implicated in various solid and hematologic malignancies, including MDS and AML, which renders them appealing targets for the development of novel therapeutic strategies. Materials: STAT3, STAT5A or STAT5B knock-downs have been generated in two AML (MOLM-13 and Kasumi-1) and two MDS (MDS92 and MDS-L, gift of Prof. Kaoru Tohyama) cell lines, using shRNAs through lenti-viral delivery. The 4 biological replicates with the highest levels of knock-downs for STAT5A, STAT5B or STAT3 have been selected for each cell line and mRNA sequencing has been performed. To define the genome-wide binding of STAT5A, STAT5B and STAT3 in the two conditions, CUT&Tag has been performed in wild type MOLM-13, Kasumi-1 and MDS-L cells. Additionally, ATAC sequencing has been carried out in the same cell lines to investigate the chromatin conformation changes between the two conditions. Furthermore, STAT3, STAT5A and STAT5B knock-downs have been conducted using shRNAs through lenti-viral delivery in CD34+ enriched cells, isolated from bone marrow mononuclear cells of 14 high-risk MDS patients and 14 secondary (MDS-related) AML patients. The samples have been processed for mRNA-Sequencing to model the leukemic transformation in primary human cells. Results: To elucidate the role of STAT3 and STAT5 in MDS to AML transformation, changes in STAT3 and STAT5 target gene networks have been identified. mRNA sequencing data from STAT3, STAT5A or STAT5B knock-downs in MDS and AML cell lines revealed a distinct role of the three factors in each state. The differential binding of each factor, explored through CUT&Tag, and the gene expression profiles in MDS and AML reciprocally reveal a different function of each factor, including pathways involved in cell cycle and apoptosis. Also, the direct and indirect target genes of STAT3, STAT5A and STAT5B in each condition, as well as a possible interplay between STAT3 and STAT5 activation in AML has been uncovered. The exploration of the chromatin accessibility landscapes between the MDS and AML conditions through ATAC sequencing has provided an insight into the chromatin conformation states, unveiling potential leukemia promoting networks, as well as the involvement of STAT3 and STAT5 pathways in the malignant transformation. Furthermore, a potential cross talk of STAT3 and STAT5 factors with the IKAROS family members, has been linked to AML transformation, as significant differential regulation has been detected between MDS and AML conditions after knock-down of STAT5A and STAT5B. The results are currently validated in primary hematopoietic cells of MDS and AML patients after STAT3, STAT5A or STAT5B knock-downs. Conclusions: Our results show a different role between the two STAT5 factors (STAT5A and STAT5B) in MDS and AML conditions and delineate the role of STAT5A, STAT5B and STAT3 factors and their target gene networks in leukemic transformation, thus providing novel targets for therapeutic management of MSD and AML. Funding: This project is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813091 (ARCH)

The Zymogen Form of Caspase-1 Is Required to Fine Tune Excessive Cell-Intrinsic Inflammation in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is characterized by the expansion of clonally-derived mutant hematopoietic stem and progenitor cells. Many patients do not respond to standard chemotherapy because AML cells acquire mutations that exploit inflammatory and cell death pathways, allowing them to evade cell death. Targeted therapies are urgently needed to block AML cell pro-survival and inflammatory signaling for more effective outcomes. One promising area of interest is Caspase-1 (CASP1), a cysteine-aspartic acid protease responsible for cleaving the precursors of interleukin-1β (IL-1β) and Gasdermin D, which induces pyroptosis, into their active mature peptides. In myeloid malignancies, IL-1β levels are significantly elevated, and pyroptosis has been linked to ineffective hematopoiesis as observed in MDS, thereby implicating CASP1. CASP1 is typically expressed as a zymogen (Pro-CASP1) that can be cleaved into an active enzyme. In this report, we present a novel and unexpected role for Pro-CASP1, which appears to be independent of its proteolytic function, in MDS/AML leukemic cells. Pyroptosis, an inflammatory cell death mechanism, is driven by CASP1-mediated osmotic lysis and IL-1b secretion. Notably, CASP1 expression is significantly higher in AML patients compared to healthy hematopoietic cells, and elevated CASP1 expression in AML patients correlates with shorter overall survival. In leukemic cells, CASP1 primarily exists in its zymogen form, lacking the ability to induce pyroptosis. It is thus unexpected that elevated CASP1 expression, which typically leads to heightened susceptibility to pyroptotic cell death, does not result in pyroptosis in leukemic cells, and the underlying mechanisms for this evasion remain unknown. To investigate Pro-CASP1's significance in AML cells, we used CRISPR-Cas9 to create Pro-CASP1 knockout (Pro-CASP1 KO) human isogenic MDS and AML cell lines (THP1 and MDSL). Pro-CASP1 KO MDS/AML cells showed reduced cell growth and leukemic colony forming potential in methylcellulose compared to isogenic WT control cells. When Pro-CASP1 KO THP1 AML cells were transplanted into immunocompromised mice, the loss of Pro-CASP1 significantly reduced leukemic cell burden and extended overall survival. We next preformed experiments to probe the mechanism of action of Pro-CASP1 in AML cells. Previous studies suggest that Pro-CASP1 can function as a scaffold to elicit downstream signaling without cleaving into its activated form. The functional defect in Pro-CASP1 KO THP1 AML cells was rescued with full-length Pro-CASP1, but not with a scaffolding mutant of CASP1, indicating a specific dependency of leukemic cells on the zymogen form of CASP1. To precisely delineate which domains of Pro-CASP1 are essential for AML, we performed a tiled CRISPR dropout screen in MDSL and THP1 cells. The results showed similar CRISPR-Knockout High Sensitivity (CKHS) domain footprints in both cell lines mapping to the N-terminal CARD and the Inter-Domain Linker (IDL) domains. These results indicate that Pro-CASP1's proteolytic function was found to be dispensable, whereas the CARD domain, a scaffolding region, was crucial for leukemogenesis. We next investigated how loss of Pro-CASP1 affects inflammatory signaling in AML cells. Deletion of Pro-CASP1 in THP1 AML cells led to excessive and constitutive activation of NF-kB as indicated by nuclear translocation and phosphorylation of p65/RelA. Restoring NF-kB regulation using the inhibitor of kappa B (IkB) super-repressor (IkB-SR) was sufficient to reverse the leukemic progenitor defect and cell viability in Pro-CASP1 KO AML cells. These findings suggest that Pro-CASP1 is required for AML cells to finetune canonical NF-kB activation. Conversely, loss of Pro-CASP1 results in excessive NF-kB activation that is incompatible with AML, consistent with previous observations (Ellegast et al., Cancer Discovery 2022). In summary, Pro-CASP1 serves as a critical signaling hub, fine-tuning NF-kB pathways independently of its proteolytic function in leukemic cells. Its loss exposes leukemic cells to functional and therapeutic vulnerabilities.

Inhibition of Topors Ubiquitin Ligase Augments the Efficacy of DNA Hypomethylating Agents through DNMT1 Stabilization

DNA hypomethylating agents (HMAs) are commonly used to treat myeloid malignancies including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, their therapeutic effects are not satisfactory. We performed CRISPR-Cas9 knockout (KO) screening to identify molecular targets that enhance the efficacy of HMAs. MDS-L and MOLM-13 cells expressing Cas9 were infected with an sgRNA lentiviral library containing 12,409 sgRNAs targeting 1,383 epigenetic factors and exposed to low-dose HMAs, decitabine (DAC) or azacitidine (AZA), for 14 days. The read counts of sgRNA against TOPORS which encodes a ubiquitin/SUMO E3 ligase, were reproducibly decreased during culture specifically in the presence of HMAs in both screening. To validate the screening results, we performed competitive growth assays by co-culturing parental cells and TOPORS-KO cells using MOLM-13, MDS-L, SKK-1, and SKM-1 cells. In all cell lines, GFP-positive TOPORS-KO cells decreased over time only in the presence of HMAs. We established single-cell clones of TOPORS-KO MOLM-13 and MDS-L cells using two different sgRNAs. The cell growth of all TOPORS-KO clones was dramatically suppressed only in the presence of DAC. Mice transplanted with TOPORS-KO cells showed significantly longer survival when treated with DAC. Next, we analyzed Topors-deficient mice and no significant changes were observed in the peripheral blood or in the percentage of bone marrow (BM) hematopoietic stem and progenitor cells. Topors−/− BM cells successfully restored hematopoiesis in lethally irradiated recipient mice at levels comparable to those of WT BM cells after transplantation, suggesting that Topors is dispensable for normal hematopoiesis. Topors−/−MLL/AF9 leukemic cells showed comparable growth in the absence of DAC but significantly inferior growth in the presence of DAC compared to WT MLL/AF9 leukemic cells. We transplanted them with support BM cells into lethally irradiated recipient mice and treated with DAC. Topors−/− leukemic cells significantly decreased during treatment compared with WT leukemic cells. Enhanced cytotoxic effects of TOPORS KO combined with HMAs was closely associated with increased apoptosis. Cell cycle analysis revealed that among TOPORS-KO cells, those in the G2/M phase accumulate and those in the S phase are reduced upon DAC treatment. RNA sequencing on cells treated with DAC showed that gene sets associated with chromosome segregation and mitosis are significantly downregulated in TOPORS-KO cells. Addback experiment revealed that TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. To identify TOPORS ubiquitination substrates, we performed second CRISPR-Cas9 screening with the same library used as the first screening, using TOPORS-KO MDS-L and MOLM-13 clone cells. Both screening showed that UHRF1-DNMT1 axis is implicated in the augmented sensitivity to DAC. Consistently, DNMT1 remained more stable in TOPORS-KO cells than WT cells after DAC exposure not only in human MDS/AML cell lines but also in murine leukemic cells. HMAs incorporated into DNA trap DNMT1 and form DNA-DNMT1 crosslinks, which causes apoptosis and mitotic defects. DNMT1-DNA crosslink formation triggers the prominent SUMOylation of DNMT1 followed by ubiquitination, which promotes the resolution of crosslinks via proteasomal degradation and cell fitness. Mass analysis of ubiquitinated peptides and immunoprecipitation with Nanobody against DNMT1 demonstrated that TOPORS loss causes accumulation of SUMOylated DNMT1 due to inefficient ubiquitination, suggesting that TOPORS works as SUMO-targeted ubiquitin ligase for DNMT1. In patient samples treated with AZA (n=23), treatment resistance significantly correlated with TOPORS expression levels. Furthermore, an ubiquitination inhibitor TAK-243 as well as a SUMOylation inhibitor TAK-981 showed synergistic effect with HMAs through DNMT1 stabilization. These results suggested that they are likely to be promising therapeutic agents in clinical practice. Our findings unveil a novel mechanism of resistance to HMAs and provide an attractive therapeutic strategy for myeloid malignancies that interferes with resolution of DNA-DNMT1 crosslinks by targeting DNMT1 post-translational modification.

Abstract 525: Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells

Abstract Therapies for acute myeloid leukemia (AML) patients are suboptimal due mainly to relapse of disease after treatment. Leukemia stem cell (LSC) populations maintain the disease and serve as a reservoir for relapse. Therefore, treatments that can eradicate LSCs will improve the outcomes of patients with AML. Our preliminary data have shown that the interleukin-1 (IL-1) pathway is upregulated following treatment and disease progression in AML and plays an important role in LSCs in vitro and in vivo. To target IL-1 signaling, we disclose the structure-activity relationship guided development of UR241-2, a novel small molecule compound of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4), which are critical downstream factors in IL-1/Toll-Like Receptor (TLR) signaling. UR241-2, at single dose (5 nM) screening exhibited high target selectivity; other than IRAK1/4 only 9 kinases in a panel of 682 kinases were inhibited by >25% activity. The NanoBRET assay using HEK293 cells transfected with targeted kinase vectors showed notably selective target engagement of UR241-2 against IRAK4 (IC50 = 6.07 nM). By using the in vitro NF-kB reporter assay and immunoblotting analysis, we identified that UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB and the phosphorylation of P65 and P38, following IL-1 stimulation. Next, we treated murine MLL-AF9 AML cells and human MDSL cells with UR241-2 and two IRAK4 inhibitors (PF-06650833 and CA-4948) that are currently in clinical or preclinical studies. We found that UR241-2 significantly inhibits colony-forming ability in AML and myelodysplastic syndromes, with similar or better efficacy compared to PF-06650833 or CA-4948. We also determined that UR241-2 suppresses LSC clonogenicity in primary human AML cells at diagnosis and in relapse while minimally impacting normal hematopoietic stem and progenitor cell function. Last, we investigated the impacts of UR241-2 on LSC engrafted ability in vivo. We treated murine MLL-AF9 cells with UR241-2 and PF-06650833 ex vivo for three days and transplanted them into recipient mice. After four weeks, we observed that UR241-2 significantly suppresses the engraftment levels of AML cells in bone marrow. It also decreases the WBC in peripheral blood and increases the RBC, HGB and PLT counts compared to the vehicle control group. Taken together, our findings demonstrated that targeting IRAK1/4 using the novel compound UR241-2 is able to impair LSC clonogenicity and engraftment capability in AML following treatment and disease progression. This provides a potential therapeutic strategy to eradicate the LSC population and improve the outcome of AML patients. Citation Format: Tzu-Chieh Ho, Rakesh K. Singh, Hiroki Kawano, Mark W. LaMere, Jian Wang, Paula M. Vertino, Nikolay V. Dokholyan, Laura M. Calvi, Craig T. Jordan, Michael W. Becker. Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 525.

CCRL2 affects the sensitivity of myelodysplastic syndrome and secondary acute myeloid leukemia cells to azacitidine.

Better understanding of the biology of resistance to DNA methyltransferase (DNMT) inhibitors is required to identify therapies that can improve their efficacy for patients with high-risk myelodysplastic syndrome (MDS). CCRL2 is an atypical chemokine receptor that is upregulated in CD34+ cells from MDS patients and induces proliferation of MDS and secondary acute myeloid leukemia (sAML) cells. In this study, we evaluated any role that CCRL2 may have in the regulation of pathways associated with poor response or resistance to DNMT inhibitors. We found that CCRL2 knockdown in TF-1 cells downregulated DNA methylation and PRC2 activity pathways and increased DNMT suppression by azacitidine in MDS/sAML cell lines (MDS92, MDS-L and TF-1). Consistently, CCRL2 deletion increased the sensitivity of these cells to azacitidine in vitro and the efficacy of azacitidine in an MDS-L xenograft model. Furthermore, CCRL2 overexpression in MDS-L and TF-1 cells decreased their sensitivity to azacitidine. Finally, CCRL2 levels were higher in CD34+ cells from MDS and MDS/myeloproliferative neoplasm patients with poor response to DNMT inhibitors. In conclusion, we demonstrated that CCRL2 modulates epigenetic regulatory pathways, particularly DNMT levels, and affects the sensitivity of MDS/sAML cells to azacitidine. These results support CCRL2 targeting as having therapeutic potential in MDS/sAML.

ASXL1 Mutations Are Associated with a Response to the Combination of Alvocidib and 5-Azacytidine in Higher-Risk Myelodysplastic Syndromes

Introduction: Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are perspective therapeutic approaches in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (MDS). Inhibitors of BCL-2 family proteins previously showed potent activity against AML and higher-risk MDS. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has previously shown clinical activity in AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and HMAs 5-Azacytidine (5-AZA) or decitabine in higher-risk MDS patients was recently completed (NCT03593915). However, biomarkers of response to 5-AZA + Alv combination are insufficiently characterized. In order to possibly identify biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-AZA effects in a cohort of n=45 higher-risk MDS patients. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of aged-matched healthy controls (HC, n=11) were obtained from bone specimen after femur endoprosthesis surgery. HSPCs were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-AZA for 48h, Alv for 24h or their sequential combination (5-AZA for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of primary MDS HSPCs was assessed using NOXA-derived T-MS1 peptide. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes. For in vivo tumor transfer experiments, splenic cells from CD45.2+Asxl1Y588X transgenic (Tg) mice were injected intravenously into sublethally irradiated CD45.1+ recipients through tail veins. Engrafted mice were treated with 1 mg/kg 5-AZA intraperitoneally (i.p.) on days 1-3 (qdx) every other week. Alv was administered i.p. at a dose of 2.5 mg/kg on day 5 (qw) in the weeks when 5-AZA was applied. Mice were sacrificed 11 weeks after the transplantation and the percentage of CD45.2+ cells in the BM was assessed using flow cytometric analysis. Results: The combination of 5-AZA+Alv showed an additive cytotoxic effect on MDS cells in CTG cell viability assays (median cell viability was 74%, 74% and 55% for 5-AZA, Alv and combination, respectively, p<0.0001). In annexin-V apoptotic assays, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to HC (median percent of apoptotic and dead cells was 37% for MDS vs 26% for HC, p=0.0256). In analogy to in vitro studies, accelerated clearance of xenografted MDS-L cells from the peripheral blood of NSGS mice was observed after 5-AZA+Alv treatment compared to single agents. MCL-1 dependency of primary MDS samples did not significantly correlate with cytotoxic effects of Alv and combination treatment. However, the presence of ASXL1 mutations was significantly associated with higher cytotoxic activity of 5-AZA+Alv combination in univariable (p=0.0044) and multivariable (p=0.042) analyses. Increased sensitivity of BM cells to the cytotoxic effects of 5-AZA+Alv was also observed in Asxl1Y588XTg mice. The combination treatment facilitated clearance of CD45.2+Asxl1Y588X mutant leukemic cells from CD45.1+ recipients compared to 5-AZA monotherapy (p=0.0156). Histological analysis of BM cells after 5-AZA+Alv treatment in CD45.1+ recipients showed decrease in the percentage of immature blasts and improved development of mature BM cells when compared to 5-AZA monotherapy (p=0.0391). Improved cytotoxic effects of Alv and 5-AZA+Alv in ASXL1 mutant compared to ASXL1 wild type MDS samples were associated with higher expression of pro-apoptotic MCL-1 antagonist NOXA (p=0.0017). Conclusions: We provided a pre-clinical rationale for the use of the 5-AZA+Alv combination for higher risks MDS and proposed ASXL1 mutations as potential genetic biomarkers of response in prospective clinical trials.

P415: TARGETED 3RD-GENERATION (NANOPORE) SEQUENCING REVEALS MANY NOVEL CIRCULAR RNAS OF THE APOPTOSIS-RELATED BCL2L12 GENE, EXPRESSED IN HUMAN CELLS FROM ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES

Background: Circular RNAs (circRNAs) are non-coding RNAs generated via back-splicing. They are indirect – yet pivotal – regulators of gene expression, as many of them bind microRNAs (miRNAs) and render them unavailable to exert their direct regulatory functions. They also interact with chromatin, altering its structure and affecting transcription of several genes. Lastly, though considered as non-coding, few of them have also been shown to encode short, functional polypeptides. Due to their multifaceted role and the deregulation of their expression in cancer, circRNAs are related to the development and progression of malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). BCL2L12 is a prominent member of the apoptosis-related BCL2 family, with an already established role in AML. Moreover, BCL2L12 mRNA expression has been associated with chemoresistance of malignant bone marrow blasts. However, the identity and role of the circRNAs produced by alternative back-splicing of BCL2L12 primary transcripts in MDS and AML has not been investigated, so far. Aims: We sought to determine the identity of BCL2L12 circRNAs expressed in MDS and AML cell lines and to explore their putative interactions with miRNAs having an established role in these disease entities. Methods: MDS-L cells [MDS with del(5q) and complex karyotype], NB-4 and HL-60 cells (acute promyelocytic leukemia with and without PML-RARA, respectively), U-937 and THP-1 cells (acute monocytic leukemia), KASUMI-1 cells (AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1) and MOLM-13 cells (AML with t(9;11)(p21.3;q23.3); KMT2A-MLLT3) were propagated. Total RNA was extracted from these seven cell lines; 2μg of each RNA extract were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of the BCL2L12 gene; thus, only cDNAs from BCL2L12 circRNAs were amplified. After having mixed and purified all nested-PCR products of each cell line cDNA, nanopore sequencing libraries were built. Following nanopore sequencing and basecalling in a MinION Mk1C 3rd-generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA identification was performed using an in-house–built, PERL-based algorithm; miRDB was used for prediction of their interactions with miRNAs. Results: We discovered 83 novel BCL2L12 circRNAs, 9 of which were common between MDS-L cells and at least one AML cell line. The exon structure of these novel circRNAs showed a remarkable diversity, comprising exons also encountered in messenger RNAs (mRNAs) of BCL2L12 as intronic regions. This phenomenon was mostly observed in MDS-L cells. Several BCL2L12 circRNAs in NB-4 and HL-60 cells exhibited complete intron retention; additionally, a novel exon was present at a remarkable frequency in NB-4 cells. Moreover, several BCL2L12 circRNAs are predicted to sponge miRNAs with a key role in AML (miR-7-5p, miR-125b-5p and miR-182-5p) and/or MDS (miR-150-5p). Summary/Conclusion: Numerous circRNAs are produced through alternative back-splicing of the primary BCL2L12 transcripts. The circRNA expression profile of this gene differs among the MDS and AML cell lines, as well as among the six AML cell lines. Intronic regions of this apoptosis-related gene are included in several circRNAs, augmenting the repertoire of miRNAs predicted to be bound by BCL2L12 transcripts. This phenomenon was more common in the MDS cell line, followed by both acute promyelocytic leukemia cell lines. Our data suggest an important regulatory role for this gene in MDS and AML subtypes.

Abstract 5435: CCRL2 affects the sensitivity of MDS and secondary AML to azacitidine

Abstract INTRODUCTION: We recently found that the atypical chemokine receptor, CCRL2 promotes the growth of MDS and secondary AML (sAML) while CCRL2 knockdown inhibits their growth. The aim of the current study is to investigate if CCRL2 regulates pathways associated with the development of resistance to hypomethylating agents (HMA), commonly used drugs in MDS and occasionally sAML. MATERIALS AND METHODS: We used lentivirus-mediated transduction of MDS92, MDS-L and TF-1 MDS/sAML cells to suppress CCRL2 expression. Two different shRNA constructs were used. We performed RNA sequencing and gene-set enrichment analysis of CCRL2 knocked down (KD) and wild-type (WT) TF-1 cells. We measured DNA methyl-transferases’ expression by western blot, CD11b and CD71 expression was measured in MDS and sAML cells to assess cell differentiation. Apoptosis was measured by Annexin V/PI staining, and clonogenicity by methylcellulose assays. CD34+ cells were sorted from bone marrow aspirates of MDS patients before the initiation of treatment with HMA by using magnetic beads and measurement of CCRL2 was performed by flow cytometry. Response to HMA in MDS patients was assessed by 6 months of treatment based on the International Working Group response criteria. RESULTS: CCRL2 KD cells demonstrated suppression of pathways associated with PRC2 complex activity, histone modification, and DNA methylation. CCRL2 KD also lead to a more prominent degradation of DNMT1, DNMT3A and DNTM3B under azacitidine treatment. CCRL2 increased apoptosis in response to 0.5 and 1 μM azacitidine (P&amp;lt;0.010) and MDS-L cells (P&amp;lt;0.010 with both sh1 and sh2). Similarly, CCRL2 knockdown increased morphologic differentiation with both 0.5 and 1 μΜ azacitidine (P&amp;lt;0.010) as well as increased the clonogenic inhibition caused by azacitidine (P&amp;lt;0.05). In order to analyze the effect CCRL2 clinically, we analyzed CCRL2 expression in CD34+ cells from patients undergoing HMA treatment. Non-responders to HMA (progressive disease) express higher levels of CCRL2 compared to CD34+ cells from responders (complete remission, partial remission or stable disease) (P=0.020). DISCUSSION: Our analysis suggests that CCRL2 regulates the expression of genes associated with induction of PRC2-mediated histone modification and DNA methylation in sAML cells. CCRL2 suppression also increased the sensitivity of MDS and sAML cells to azacitidine. In addition, increased CCRL2 expression in MDS cells is associated with worse response to HMA. These data suggest that targeting CCRL2 has therapeutic potential in MDS and sAML. Citation Format: Theodoros Karantanos, Patric Teodorescu, Brandy Perkins, Marios Arvanitis, Ilias Christodolou, Christopher Esteb, W. Brian Dalton, Tania Jain, Amy E. DeZern, Lukasz P. Gondek, Mark J. Levis, Gabriel Ghiaur, Richard J. Jones. CCRL2 affects the sensitivity of MDS and secondary AML to azacitidine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5435.

Effects of Rigosertib (RIGO) Alone or in Combination with Azacitidine or Vorinostat on Epigenetic Reprogramming of CD34+ Cells in the Myelodysplastic Syndrome

Rigosertib (RIG) which functions as a RAS-mimetic, binds to the RAS-binding domain (RBD) impacting many pathways that involve RBD-mediated interactions, including the RAS-Raf and AKT/PI3-kinase pathways (Divakar et al., Cell 2016). Recently, we demonstrated that RIG also functions as an epigenetic modifier in MDS. RIG alone or in combination with Azacitidine (AZA) exerted epigenetic effects on the global histone post-translational modifications (PTMs), chromatin remodelers, HDACs, and DNMTs and caused enhanced apoptosis in MDS (MDS-L) and AML (BW-90) cell lines as well as in bone marrow cells (BM) derived from pts with MDS (Chaurasia et al ASCO 2016). In clinical trials, the combination of RIG and AZA demonstrated an overall response rate of 76% in all MDS patients; 62% response in pts who had failed prior hypomethyating agent (HMA) (Navada et al ASH 2016) and 85% in HMA naive pts. The growing evidence suggests that combined therapy may be more effective than either agent alone. To further study these effects, we explored activity of RIG in combination with Vorinostat (VOR), a histone deacetylase inhibitor, on the cell lines. Combined treatment in sequential order VOR/RIG and RIG/VOR potentiates far greater effects on the global PTMs, HDACs, DNMTs, chromatin remodelers, cell cycle checkpoint proteins and PI3/AKT pathway than RIG/AZA or AZA/RIG.RIG in combination with AZA or VOR (AZA/RIG, RIG/AZA, VOR/RIG or RIG/VOR) demonstrated differential effects on the association of RNA polymerase II (Pol II) with active histone marks (H3K4me3 and H3K4me2) in MDS-L and BW-90 cells. An overall decrease in association of Pol II/H3K4me2 was observed with the combinations (AZA/RIG, VOR/RIG or vice versa) compared to control in MDS-L and BW-90, 10-33% (ANOVA, p=0.0006), 9-20% (ANOVA, p=0.0004), respectively. Significant differences were observed in association of H3K4me3/Pol II in BW-90 cells (7-30%; ANOVA, p<0.0001). Similarly, in BM from a pt with MDS after 1 cycle of RIG and AZA treatment demonstrated a decrease in association of Pol II with H3K4me2 (67%) and H3K4me3 (28%).Treatment of MDS-L cells with RIG alone or in combination with AZA failed to induce expansion of CD34+ cells, but the RIG/VOR combination induced 1.9-fold expansion of CD34+ cells (ANOVA p=0.002). Individually AZA or VOR treatment led to an enhanced expansion of CD34+ cells, 3.8 and 4.0 fold, respectively (ANOVA, p=0.004). RIG alone or with AZA yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSPCs) (ANOVA, p=0.006). However, a marked decrease in ALDH activity was observed in AZA or VOR or RIG/VOR, that was inversely proportion to the expansion of CD34+ cells.In a single MDS pt treated with RIG/AZA, expansion of primitive HPSCs expressing low levels of CD34 was observed. A highly expressed subpopulation of HPSCs, which co-existed prior to treatment of the pt and disappeared upon treatment, suggests either an induced cell death or commitment of HSPCs towards improved lineage differentiation in conjunction with clinical response.In order to evaluate effects of RIG on expansion of CD34+ cells, we examined the pluripotent genes (SOX2, OCT4 NANOG and ZIC3) in the BM from MDS pts, prior and after RIG/AZA treatment. RNA expression levels of SOX2, OCT4, NANOG and ZIC3 were elevated post treatment (≥2 fold). In MDS-L cells the pluripotency genes were highly upregulated in the presence of RIG or RIG/AZA or RIG/VOR (1.7-34 folds). These findings indicate that expression of pluripotency genes is a consequence of epigenetic reprogramming that favors expansion of more primitive HSPCs in a pt, while increase in ALDH activity was inversely proportional to the enhanced expansion of CD34+ cells in MDS-L suggesting a more primitive state of HSPC.The epigenetic events modulated by RIG in combination with either AZA or VOR led to global histone PTMs, differential Pol II association with active histone marks, epigenetic reprogramming of pluripotency genes and expansion of primitive HSPC with downregulation of the PI3K/AKT pathway and cell cycle checkpoint proteins. Collectively, these studies indicate that epigenetic effects of RIG on chromatin alterations lead to HSPC reprogramming that may lead to improved hematopoietic function and response in the clinical setting. These preclinical models suggest potential novel clinical strategies to improve outcomes for patients with higher-risk MDS. DisclosuresReddy:Onconova Therapeutics: Equity Ownership.

Activation of SIRT1 Deacetylase As a Therapeutic Approach for Myelodysplastic Syndromes By Restoring TET2 Function

Myelodysplastic syndromes (MDS) which arise from a primitive CD34+ hematopoitic stem/progenitor cell (HSPC), are incurable by non-transplant therapy. Better understanding of MDS HSPC regulation is critical for developing targeted therapies against the fatal disease. Tet methylcytosine dioxygenase 2 (TET2), initiating DNA demethylation through converting 5-methylcytosine to 5-hydromethylcytosine (5hmC), is the most frequently mutated gene in MDS. TET2 alterations lead to DNA hypermethylation and dysregulated gene expression in HSPCs, resulting in enhanced self-renewal and skewed myeloid specific expansion. While TET2's biological function has been well studied, the knowledge of its regulation is limited. We had demonstrated the TET2 c-terminus acetylation was reversed by SIRT1 deacetylase which maintains normal HSPCs activity under stress. We also observed that SIRT1 protein in MDS CD34+CD38- was expressed below normal levels, and that knockdown (KD) of SIRT1 enhanced in vivo growth of MDS-L cells, a patient derived MDS cell line, is associated with TET2 acetylation (Blood. 2016;128:1053). These results indicate an important role for SIRT1 in MDS HSPCs maintenance.To further assess SIRT1 function in MDS, we ectopically expressed SIRT1 in primary MDS CD34+ cells enriched for HSPCs and MDS-L cells, respectively. Overexpression (OE) of SIRT1 resulted in inhibition of colony formation of MDS CD34+ cells (p<0.01, n=3) but not normal counterpart (p>0.05, n=3). SIRT1 OE also suppressed MDSL colony formation (p<0.01) and cell growth (p<0.01). SIRT1 OE significantly reduced MDS-L cell BM engraftment in NSGs mice relative to controls (human CD45+ cells: 5.7±0.8% of BM in SIRT1 OE group vs 31.4 ± 5.8% in control, p<0.01, n=7), resulting in delayed mortality of recipient mice (median survival: 52 days vs 35 days; p<0.05, n=5). Meanwhile, treatment of SIRT1 agonist SRT1720, reduced colony formation of primary MDS CD34+ cells in a dose dependent manner (p<0.01, n=9). Similar results were also achieved in SRT1720 treated MDS-L cells. Furthermore, TET2 was hyperacetylated in primary MDS cells (n=3) compared to normal counterpart (n=3). Next, we tested whether inhibition of MDS HSPCs following SIRT1 activation was related to TET2 activation via protein deacetylation. Indeed, enhanced TET2 activity marked by 5hmC was observed in MDS CD34+ cells upon SRT1720 treatment (P<0.05, n=3). TET2 KD diminished growth inhibition induced by SIRT1 OE or SRT1720, indicating a requirement of TET2 for SIRT1 function. Through mass spectrometry analysis, three conserved acetylated lysine residuals (K1472, K1473, K1478) were identified in TET2 catalytic domain (CD). Engineered lysines to glutamines (TET2 3KQ) mutant, mimicking acetylated TET2, produced lower 5hmC, and led to increased colony growth compared to MDS-L expressing TET2 WT (p<0.01). We further evaluated the effects of SRT1720 using NUP98-HOXD13 (NHD13)MDS mouse. This murine MDS is transferable and secondary recipients receiving primary NHD13 MDS cells succumb to MDS at 4 months, allowing generation of a cohort of MDS mice for preclinical studies of therapeutics against MDS. CD45.2+ BM cells from donor NHD13+ mice showed evidence of dysplasia were harvested, and transplanted into preconditioned congenic recipients expressing CD45.1. SRT1720 treatment (100mg/kg/day for 12 weeks, p.o.) resulted in partial reversal of MDS-like disease developed in recipients receiving MDS cells, including an increase in HGB, PLT, RBC and WBC (p<0.01,n=6). Importantly, significant reduction of NHD13+ CD45.2+ chimerism in LSK and other progenitors from BM were also observed (p<0.01, n=6). As expected, 5hmC level in lineage depleted cells from SRT1720 treated mice was increased (p<0.01). Secondary transplantation was performed to evaluate the effects of SRT1720 on MDS initiating cells. We observed that the mice receiving cells from SRT1720 treated donors showed reduced NHD13+ CD45.2+ cell long-term engraftment (p<0.01, n=5).In summary, we demonstrated that genetic overexpression or pharmacological activation of SIRT1 drastically inhibited MDS maintenance, and that TET2 function was required for the inhibitory effect of SIRT1. These results support the hypothesis that SIRT1 deficiency promotes MDS cell expansion through TET2 inactivation and support the rationale for exploring SIRT1 activation as a promising approach to selectively target MDS stem cells. DisclosuresNo relevant conflicts of interest to declare.

A CRISPR RNA-Binding Protein Screen Reveals Novel Regulators of RUNX1 Alternative Polyadenylation

Alternative polyadenylation (APA) is a crucial post-transcriptional process that regulates the 3' end formation of over 75% of all human genes. Despite its prevalence and importance during development and in disease, there are few studies that focus on APA in the hematology field. Interestingly, APA affects the coding sequence of RUNX1, a pivotal transcription factor for proper hematopoiesis. Usage of the most proximal RUNX1 polyadenylation sequence (PAS) in upstream alternative terminal exon 7a results in the C-terminally truncated RUNX1a isoform. RUNX1a uniquely promotes self-renewal and expansion of hematopoietic stem cells (HSCs). In the endogenous context, usage of this proximal PAS is tightly restricted, resulting in polyadenylation at one of three distal PAS in exon 8 and producing the RUNX1b/c isoforms. RUNX1b/c are much more abundant and promote HSC differentiation and lineage commitment. Importantly, the ratio of RUNX1a to RUNX1b/c is developmentally-regulated. Therefore, it is highly significant to uncover the APA mechanism that controls the RUNX1 isoform ratio and consequently the balance of HSC self-renewal and differentiation. We hypothesize that there are enhancers and suppressors of the proximal RUNX1 PAS, tightly controlling the relative amount of RUNX1a produced during hematopoiesis.To identify RUNX1 APA regulators, we first developed a strategy to monitor RUNX1 proximal PAS usage in vitro. We cloned alternative terminal exon 7a, which contains the proximal PAS, along with 500 bp of up and downstream flanking intron between the two exons of a split GFP reporter. When we introduce this fluorescent minigene reporter into blood cells, two transcripts can be produced: (1) the first GFP exon splices and polyadenylates RUNX1 exon 7a, producing no GFP (RUNX1a formation) or (2) the two GFP exons are spliced together, skipping the proximal PAS in exon 7a, producing GFP (RUNX1b/c formation). Since RUNX1b/c are much more highly expressed than RUNX1a throughout hematopoiesis, we observed a robust GFP signal when this reporter was transfected into MDS-L, K562, and U937 cell lines. RT-PCR analysis confirmed that both transcripts are produced, albeit at dramatically different levels. We can thus use GFP fluorescence as a read-out for RUNX1 proximal PAS usage.Next, we generated a stable, clonal MDS-L cell line expressing this RUNX1 exon 7a split GFP reporter. We used this reporter line to perform a CRISPR screen using an sgRNA library that is enriched for targeting RNA-binding proteins (RBPs). We infected cells with the library, cultured in puromycin to force sgRNA and Cas9 integration, waited three weeks to ensure gene knockout, and then used fluorescence-activated cell sorting (FACS) to isolate GFP low and high cell populations. GFP low cells contained sgRNAs targeting RUNX1a suppressors since these cells exhibited enhanced proximal PAS usage. Conversely, GFP high cells contained sgRNAs targeting RUNX1a enhancers since proximal PAS usage was further diminished. We identified 44 putative RUNX1a suppressors and 41 putative enhancers (p < 0.05).We then sought to validate the genes revealed in our CRISPR screen by using individual sgRNAs targeting a subset of the candidates. One confirmed suppressor is HNRNPA1, an RBP which is upregulated and leukemogenic in BCR/ABL chronic myelogenous leukemia (CML). In HNRNPA1 knockout cells, there was a 5-fold increase in relative RUNX1a and a corresponding decrease in RUNX1b/c levels by RT-qPCR. This effect was dose-dependent as heterozygous HNRNPA1 cells exhibited an intermediate 2.5-fold increase in RUNX1a mRNA. We verified this data by performing shRNA knockdown of HNRNPA1 in MDS-L and K562 cells, again observing a significant increase in relative RUNX1a levels. Interestingly, HNRNPA1 is highly expressed throughout the course of hematopoiesis, indicating that a cooperating protein helps facilitate this suppressive effect. Indeed, numerous HNRNPA1-interacting proteins were also identified in our screen and are the subject of current studies.In summary, we devised a fluorescent minigene reporter to monitor RUNX1 APA in vitro. Using this reporter, we performed a CRISPR RBP screen and identified HNRNPA1 as the founding member of a complex that suppresses RUNX1a production during hematopoiesis. HNRNPA1 and additional regulators represent novel proteins that have the potential to regulate HSC behavior by modifying the RUNX1 isoform ratio. DisclosuresNo relevant conflicts of interest to declare.

Decitabine Induces Change of Biological Traits in Myelodysplastic Syndromes via FOXO1 Activation.

Decitabine (DAC) is considered to be a profound global DNA demethylation, which can induce the re-expression of silenced tumor suppressor genes. Little is known about the function of tumor suppressor gene FOXO1 in myelodysplastic syndromes (MDS). To address this issue, the study firstly investigated differentially expressed genes (DEGs) for DAC treatment in MDS cell lines, then explored the role of FOXO1 through silencing its expression before DAC treatment in MDS. The results showed that FOXO1 exists in a hyperphosphorylated, inactive form in MDS-L cells. DAC treatment both induces FOXO1 expression and reactivates the protein in its low phosphorylation level. Additionally, the results also demonstrated that this FOXO1 activation is responsible for the DAC-induced apoptosis, cell cycle arrest, antigen differentiation, and immunoregulation in MDS-L cells. We also demonstrated DAC-induced FOXO1 activation upregulates anti-tumor immune response in higher-risk MDS specimens. Collectively, these results suggest that DAC induces FOXO1 activation, which plays an important role in anti-MDS tumors.

Efficacy of Tubulin Polymerization Inhibitor in Myelodysplastic Syndrome

Background: Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Tubulin belongs to protein superfamily of globular proteins. Monomeric tubulin can polymerize into microtubules, which play an important role in the attachment and segregation of chromosomes in various phases of cell division. Therefore, the targeting of microtubules represents a therapeutic strategy against both solid and hematological cancers. The first approved tubulin binding agent by the FDA was vincristine, which has been clinically used to treat multiple types of cancers, particularly hematological malignancies. Over the past few decades, additional tubulin binding agents have been developed and received FDA approval, mostly for applications to cancer therapy. These agents have been classified by their binding sites on tubulin, which influences their roles in the inhibition or stabilization of polymerized microtubules. We herein investigated the efficacy of PTC-028, a novel microtubule polymerization inhibitor for MDS. Method: Anti-MDS efficacy of PTC-028 was studied using human MDS cell lines and primary MDS cells in vitro. The efficacy of PTC-028 was also assessed in a xenograft mouse model of MDS using an MDS cell lines. PTC-028 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometry and RNA sequencing. Results: A previous study reported that PTC596 suppressed cell proliferation and induced apoptosis in AML cell lines. Since MDS is regarded as a pre-leukemic stage, we examined the effects of PTC-028, another novel microtubule polymerization inhibitor, on MDS cells. PTC-028 induced a dose-dependent inhibition of cell proliferation on MDS cell lines, such as MDS-L and SKM-1 cells. Caspase 3/7 activity was also significantly induced in MDS-L and SKM-1 cells in the presence of PTC-028, suggesting the induction of apoptotic cell death by PTC-028. We then isolated CD34+ cells from primary MDS BM samples and investigated the efficacy of PTC-028 on primary MDS cells. The efficacy of PTC-028 in CD34+ MDS cells was also confirmed by cell proliferation assays. To enhance the therapeutic benefit of PTC-028 on MDS cells, we investigated synergism between PTC-028 and DNA hypomethylating agents. We treated MDS-L and SKM-1 cells with PTC-028 in combination with DNA hypomethylating agents. After 3 days of culture, cell growth was analyzed by MTS and Annexin V assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit the growth and induce apoptosis of MDS cells. We assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using an MDS cell line. Recipient mice were treated with PTC-028 for 7 weeks. PTC-028 significantly inhibited the growth of MDS-L cells and prolonged the overall survival of recipient mice. Furthermore, a significant synergistic effect was observed between PTC-028 and decitabine. Mice that received combination therapy showed moderate weight loss 11 days after the initiation of the treatment, but subsequently recovered. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We then investigated the effect of PTC-028 on the levels of soluble (unpolymerized) versus polymerized tubulin in MDS-L cells. Cells were treated with PTC-028 and paclitaxel for 4 hours, and cell lysates were then separated into soluble and polymerized fractions by centrifugation. The visualization of tubulin fractions by Western blotting demonstrated that the PTC-028 treatment for 4 hours resulted in the near-complete loss of polymerized microtubules. In contrast, polymerized microtubules increased in cells treated with paclitaxel, which stabilizes microtubules against depolymerization. These result indicates that PTC-028 also acts as a microtubule polymerization inhibitor. Conclusion: Our data reveal a possible chemotherapeutic strategy for PTC-028 and PTC-596 in MDS by disruption of microtubule dynamics as a single agent and in combination with hypomethylating agents. The present study provides a preclinical framework for the clinical evaluation of this promising therapeutic approach to improve outcomes in MDS patients. Disclosures Lennox: PTC therapeutics: Current Employment. Sheedy:PTC therapeutics: Current Employment. Weetall:PTC Therapeutic: Current Employment.

Rigosertib in Combination with Azacitidine Impacts Metabolic and Differentiation Pathways in the MDS-L Cell Line

Background Myelodysplastic Syndrome (MDS) is characterized by ineffective clonal hematopoiesis with peripheral blood cytopenias, leading to death from infection or bleeding. Azacitidine (AZA), a hypomethylating agent (HMA) is the standard of care for treatment of MDS patients (pts) with higher-risk MDS [Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017]. Responses to AZA occur in 50% of pts with significant effects on hematopoiesis ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence [Silverman LR, et al. Leukemia, 1993]. AZA treatment is associated with global DNA hypomethylation, including human endogenous retroviruses (HEV) which further activates innate immune signaling [Chiappinelli KB,et al. Cell, 2015]. The exact mechanism by which AZA improves hematopoiesis is unknown. AZA improves overall survival of pts, yet despite this, 100% of pts ultimately fail treatment with worsening cytopenias or transformation to leukemia [Silverman LR, et al. B. J Clin Oncol, 2002; Cancer, 2011]. Thus, understanding the mechanism of resistance and identification of targets which can reverse HMA failure and improve hematopoiesis in MDS pts is critical. Our clinical data demonstrate that AZA combined with Rigosertib (RIGO), a novel Ras mimetic that inhibits Ras/Raf signaling [Athuluri-Divakar SK, et al. Cell, 2016], yields a response rate of 54% of pts who were HMA failures [Navada SC, et al. EHA 2019]. The response was associated with significant improvement in hematopoiesis and represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The precise mechanism that leads to reversal of the resistance phenotype is poorly understood. Methods: We investigated the differential protein expression in response to different treatment (AZA, RIGO alone and sequential combinations RIGO/AZA; AZA/RIGO) in vitro in MDS-L cell line by Reverse phase protein array (RPPA). Further, we also studied the functional role of these treatments on differentiation in the cell line by growing cells on semi-solid media as well as by flow cytometry using various stem cell and differentiation markers (CD34, CD38, CD45, CD123). Results: RPPA analysis indicated a discrete responses to treatment in the MDS-L cell line. The response was prominent with differential expression of 43 proteins specifically in MDS-L cells treated with RIGO/AZA that include PIK3R1, AKT1, mTOR, p38 MAPK, PTEN, RPS6KA1 (Fold change (FC) &amp;lt; -2). All of these proteins are downregulated which suggests inhibition of PI3K and mTOR signaling. Proteins belonging to metabolic pathways, including ACC1 and ACLY, were found to be downregulated, whereas proteins related to mitochondrial function and oxidative phosphorylation (OXPHOS) were upregulated (FC &amp;gt; 1.5/&amp;lt; -1.5) in cells treated with RIGO/AZA. In addition, we found that MDS-L cells represented both CD34+CD38+ and CD34+CD38- populations by FACS analysis (Fig 1). AZA increases the percentage of CD34+CD38+, indicative of differentiation, whereas RIGO alone increased the percentage of CD34+CD38- cells, representing a primitive stem cell population (Table in Fig 1). Based on the combination of differentiation markers (illustrated in fig 1), we observed that RIGO alone, and sequenced as combination RIGO/AZA, impacts different progenitors such as granulocyte-macrophage progenitor (GMP), megakaryocyte erythroid progenitor (MEP), and multipotent progenitor (MPP). RIGO/AZA treatment showed a decrease in GMP, while the % of MEP was increased, as compared to other treatments. Moreover, we found a remarkable reduction in the number of colony forming units on differentiation media in response to RIGO (83%) and RIGO/AZA (90%). Conclusions: These data demonstrate that in addition to inhibition of the PI3/AKT/mTOR pathway, the RIGO/AZA combination also impacts metabolic and differentiation pathways of MDS-L cells. RIGO alone appears to promote maintenance of a primitive stem cell population, while the RIGO/AZA sequenced combination appears to push the cells toward a cycling stage with increased expression of genes associated with OXPHOS. In comparison, when treated with RIGO, cells remain in a less differentiated stage. Further studies are underway to determine the effect of metabolic changes on differentiation and maintenance of hematopoietic stem cells. Figure Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Reddy:Onconova Therapeutics Inc: Research Funding. Silverman:Celgene: Research Funding; Medimmune: Research Funding; Onconova Therapeutics Inc: Patents &amp; Royalties, Research Funding.

Role of Oligoadenylate Synthetases in Myeloid Neoplasia

Acute myeloid leukemias (AML), the most lethal forms of blood cancer, are genomic instability disorders primarily driven by somatic mutations that greatly impact proliferation and survival of mutant clones1. The multistep mechanism of disease progression can be attributed, in part, to the defects in one or more pathways involving responses to, or repair of, damaged DNA (DD) and base excision repair (BER). The basal levels of DD and BER are higher in myeloid leukemias due to higher levels of reactive oxygen species (ROS)-mediated accumulation of 8-oxyguanine (8-OG)2. Poly(ADP) Ribosylation (PARylation) by PAR polymerases (PARPs) is one of the early key steps in sensing and repairing ROS-induced DNA damage response (DDR) 3. Consistent with recent findings4,5 here we report that 2'-5'oligoadenylate synthases (OASs), a family of latent 2'-5'-adenylyl transferases, otherwise involved in cellular antiviral responses6, are also involved in PAR remodeling of the DDR in MDS and AML cells. The 2'-hydroxyl group of PAR ribose acts as an acceptor for 2'AMP in the OAS enzymatic reaction and terminates chain elongation (Fig. A). OAS1 over expression can increase genomic instability by increasing the flux of PARP-mediated DNA repair, promoting cellular proliferation (Fig. B)5. Expression analysis of OASs in AML patients (n=451, Beat AML) showed that OAS1 is upregulated (2-fold) in AML patients compared to normal bone marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPC)(Fig. C). Knockout of OAS1 and OAS2 using Crispr-Cas9 in MDS-L cells (Fig. D), a cell line derived from an MDS patient, confers sensitivity to H2O2-induced DNA damage-mediated cell death; however, OAS3 has no effect. Conversely, ectopic over expression of OAS1 or OAS2 but not OAS3 in HEK293 cells provides protection against H2O2-induced cell death (Fig. E-F). Proteomic analysis of the OAS1 and OAS2 interactome using LCMS/MS suggests that over expression of OAS1 and OAS2 perturbs the differentiation program of HSPCs that may result in neoplastic evolution. Thus, OASs modify PAR chains, promoting speedy DNA repair and cell survival along with the induction of a differentiation block in HSPCs that leads to clonal expansion. In summary, an overburdened DDR may contribute to AML pathogenesis. Therefore, inhibiting this stimulator of BER/DDR can provide a novel therapeutic avenue in myeloid neoplasms. The current study points to the probable utility of a novel therapeutic approach of targeting OAS in combination with DNA damaging agents to prevent relapse and resistance in the treatment of leukemias.

FBXO11 is a candidate tumor suppressor in the leukemic transformation of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.

Abstract 646: CRISPR/Cas9 screening to identify mechanisms of azacitidine failure in myelodysplastic syndrome

Abstract Myelodysplastic syndrome (MDS) is a heterogenous myeloid lineage malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of secondary transformation to acute myeloid leukemia (sAML). The mainstay of treatment in patients with higher-risk MDS are the hypomethylating agents (HMAs) azacitidine and decitabine, which extend overall survival by approximately 12 to 18 months. Unfortunately, disease relapse and HMA resistance are inevitable and carry a very poor prognosis. Genomic sequencing of large MDS cohorts has led to the identification of recurrent genetic abnormalities in MDS, some of which predict sensitivity to HMA therapy. For example, abnormalities in TET2, but not ASXL1, are associated with a favorable response to the HMAs. However, it is unknown why some patients with otherwise similar clinical features have variable responses to HMA therapy. We hypothesize that there are common cellular mechanisms promoting resistance to HMA therapy and that these can be therapeutically targeted to enhance treatment responses. To uncover cellular processes promoting HMA treatment failure we performed duplicate genome-wide CRISPR/Cas9 screen in the human MDS-L cell line using resistance to azacitidine as a positive selection readout. MDS-L was established from bone marrow mononuclear cells in a 52-year-old male patient with high-risk MDS. We transduced the Brunello sgRNA CRISPR library into Cas9 expressing MDS-L cells and subjected the cells to azacitidine treatment for 7 days. Cells surviving azacitidine treatment were then expanded and harvested for genomic DNA. High throughput sequencing of the barcoded DNA produced raw reads that were analyzed using the PinAPL-Py web-based software. sgRNAs appearing in duplicate with over 10-fold enrichment compared to the control library were considered significant. We identified 125 candidate genes involved in HMA resistance from the CRISPR-Cas9 screen. Pathway analysis revealed a significant enrichment of genes involved in lipid metabolism, DNA methylation, and response to small molecule catabolism. Future experiments to validate these candidates as well as test whether they contribute in vivo to HMA resistance are underway. We conclude genome-wide CRISPR-Cas9 screens for drug-resistance in the MDS-L model are feasible and may uncover future therapeutic targets to augment HMA therapy or utilize upon HMA failure. Citation Format: Michael Schieber, John D. Crispino. CRISPR/Cas9 screening to identify mechanisms of azacitidine failure in myelodysplastic syndrome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 646.

Cyclosporine enhances the sensitivity to lenalidomide in MDS/AML in vitro

Our previous study revealed that expression of G protein-coupled receptor 68 (GPR68) was upregulated in MDSL cells, a cell line representing myelodysplastic syndromes (MDS), in response to lenalidomide (LEN), and mediated a calcium/calpain proapoptotic pathway. Isx, a GPR68 agonist, enhanced the sensitivity to LEN in MDSL cells. The fact that Isx is not a U.S. Food and Drug Administration-approved drug prompts us to look for alternative candidates that could enhance the sensitivity of LEN in MDS as well as other hematologic malignancies, such as acute myeloid leukemia (AML). In the study described here, we found that regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of calcineurin (CaN), was upregulated in MDSL cells in response to LEN, possibly through degradation of IKZF1. Consistently, cyclosporin (Cys), a pharmacological inhibitor of CaN, inhibited the activity of CaN and induced apoptosis in MDSL cells, indicating that CaN provided a prosurvival signal in MDSL cells. In addition, Cys enhanced the cytotoxic effect of LEN in MDS/AML cell lines as well as primary bone marrow cells from MDS patients and AML patient-derived xenograft models. Intriguingly, pretreatment with LEN reversed the suppressive effect of Cys on T-cell activation. Our study suggests a novel mechanism of action of LEN in mediating cytotoxicity in MDS/AML via upregulation of RCAN1, thus inhibiting the CaN prosurvival pathway. Our study also suggests that Cys enhances the sensitivity to LEN in MDS/AML cells without compromising T-cell activation.

Myeloid cell–targeted miR-146a mimic inhibits NF-κB–driven inflammation and leukemia progression in vivo

AbstractNF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell–selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a–deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell–induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB–related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell–targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.