32Dcl3 Cells Research Articles

C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription

BackgroundAcute myeloid leukemia (AML) with biallelic (CEBPAbi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs.MethodsHuman AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents.ResultsWe found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo.ConclusionOur results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPAbi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers.

Loss of EFL1 Which Causes a Shwachman-Diamond Syndrome Reduces Cell Proliferation and Alters Transcriptional Profiling during Neutrophil Differentiation

Introduction. Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. Patients with SDS are predisposed to develop myeloid malignancy. Almost all individuals with SDS harbor biallelic mutations in SBDS. The partner of SBDS is EFL1, a ribosomal protein with GTPase activity. More than a dozen patients have been found with SDS due to biallelic mutations in EFL1. EFL1 serves to release EIF6, facilitating the assembly of the 80S ribosome from 60S and 40S ribosome subunits. Our lab has generated both sbds and efl1 null zebrafish, which phenocopy much of the human syndrome. We recently reported that Sbds and Efl1 depletion leads to upregulation of tp53 and cdkn1a ( p21) and accumulation of Eif6 in SDS zebrafish models, which is hypothesized to cause bone marrow failure. However, the mechanism(s) of EFL1's loss-of-function leading to neutropenia is not known. Methods. We used CRISPR/Cas9 editing to generate mutations in exon 17 of Efl1 of 32Dcl3, an IL-3-dependent murine myeloid cell line that can be differentiated to mature neutrophils. We analyzed the effects of Efl1 mutations on cell proliferation, survival, and differentiation with G-CSF. Immunoblotting and qPCR-based gene expression analysis were performed to identify affected mechanistic pathways. Results. Two clones were isolated and were sequenced to verify biallelic deleterious mutations. A clone with compound heterozygous inframe deletion and frameshift mutations showed trace expression of EFL1 (EFL1-KD) and the other with compound heterozygous frameshift mutations showed no expression (EFL1-KO). They presented a myeloblast-like morphology indistinguishable from parental cells. They proliferated significantly less than parental cells, although there was no increased apoptosis. Both EFL1-KD and KO cells showed significantly increased protein expression of EIF6 and transcriptional expression of Trp53 and Cdkn1a. EFL1 was downregulated during differentiation of parental 32Dcl3 cells to neutrophils, whereas EIF6 levels did not change. Withdrawal and replenishment of IL-3 from culturing media did not alter EFL1 protein expression. When EFL1-KD and KO were cultured in differentiating media, they did not proliferate and underwent greater apoptosis, particularly after 3 days of differentiation compared to parental 32Dcl3. The surviving EFL1-KD and KO matured into neutrophils after 7 days. Gene profiling for neutrophil differentiation identified equivalent expression of key transcriptional factors ( Cebpa, Cebpe, and Spi1) of EFL1-KD and KO in the maintenance media, except for Gfi1 which was two-fold and three-fold high in EFL1-KD and KO compared to parental cells, respectively. G-CSF induced significantly higher expression of Cebpa and lower expression of Cebpe in EFL1-KO from those in parental cells, respectively. Based on the observation that somatic genetic rescue by EIF6 mutations occurs in human SDS patients and animal models, we transfected 32Dcl3 cells with siRNA against Eif6. In parental 32Dcl3 cells, reduction of EIF6 caused slow cell growth; however, transfection of siRNA to Eif6 partially rescued proliferation of EFL1-KD and KO in both maintenance and differentiating media. Conclusions. Our results suggested that imbalance of late maturing factors of ribosome associated with SDS may cause decreased proliferation in myeloid-committed cells, thereby leading to neutropenia in SDS. Newly established Efl1-deficient cells recapitulated key observation from SDS patients, with an increased proapoptotic profile in hematopoietic cells. Because EFL1 has only been recognized recently as a rare cause of SDS, we do not know if EFL1 deficiency serves as a leukemia predisposition syndrome. We speculate that the aberrant normal neutrophil differentiation may form the foundation for myeloid neoplasia in SDS.

Sequential control of myeloid cell proliferation and differentiation by cytokine receptor-based chimeric antigen receptors.

As chimeric antigen receptor (CAR)-T cell therapy has been recently applied in clinics, controlling the fate of blood cells is increasingly important for curing blood disorders. In this study, we aim to construct proliferation-inducing and differentiation-inducing CARs (piCAR and diCAR) with two different antigen specificities and express them simultaneously on the cell surface. Since the two antigens are non-cross-reactive and exclusively activate piCAR or diCAR, sequential induction from cell proliferation to differentiation could be controlled by switching the antigens added in the culture medium. To demonstrate this notion, a murine myeloid progenitor cell line 32Dcl3, which proliferates in an IL-3-dependent manner and differentiates into granulocytes when cultured in the presence of G-CSF, is chosen as a model. To mimic the cell fate control of 32Dcl3 cells, IL-3R-based piCAR and G-CSFR-based diCAR are rationally designed and co-expressed in 32Dcl3 cells to evaluate the proliferation- and differentiation-inducing functions. Consequently, the sequential induction from proliferation to differentiation with switching the cytokine from IL-3 to G-CSF is successfully replaced by switching the antigen from one to another in the CARs-co-expressing cells. Thus, piCAR and diCAR may become a platform technology for sequentially controlling proliferation and differentiation of various cell types that need to be produced in cell and gene therapies.

Cooperative Inhibitory Effect of IDH1 Mutation and ASXL1 Mutation on Normal Myeloid Differentiation

Somatic mutations in the additional sex comb-like 1, ASXL1, have been described in various types of myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia (AML). In addition, ASXL1 mutations are frequently found in healthy population, which predispose them to not only myeloid malignancies, but also cardiovascular diseases. This condition is known as clonal hematopoiesis with intermediate potential (CHIP), although the full etiology of this preleukemic condition is not elucidated. As for AML, patients with ASXL1 mutations are known to have very poor prognosis compared to non-mutated patients. Although previous studies demonstrated that mutant ASXL1 promoted myeloid leukemogenesis via both loss-of-function and gain-of-function manners, mechanisms how ASXL1 mutations induce myeloid transformation is not fully elucidated. We analyzed publicly available datasets of AML patients, and found that isocitrate dehydrogenase 1 (IDH1) is co-mutated in 13% of AML with ASXL1. As the mechanism by which ASXL1 and IDH1 mutations cooperate to promote AML is not understood, we generated a model of AML with ASXL1 and IDH1 mutations using 32D-cl3 cells and discovered that mutant IDH1 could block myeloid differentiation only when mutant ASXL1 was present. At first, in our experiments, we retrovirally overexpressed mutant (G646WfsX12) or normal form of ASXL1 in 32D-cl3, which is a commonly used IL-3 dependent cell-line of murine myeloid precursor cell. We found that Gr.1 expression levels were decreased in these cells compared to 32D-cl3 with mutant ASXL1 (11.1% vs 26.5%, respectively; p=0.014), and that the proportion of cells in which Gr1 expression was induced by treatment with G-CSF was significantly lower (29.1% vs 54.8%, respectively; p=0.003). Next, we generated a tetracycline-inducible mutant (R132H) form of IDH1 lentiviral vector, and transfected them to these 32D-cl3 cells. We found that tetracycline-inducible mutant IDH1 inhibited G-CSF induced Gr.1 expression in the presence of mutant ASXL1 (7.1% with doxycycline vs 29.9% without doxycycline, respectively; p<0.01), while in the absence of mutant ASXL1, forced expression of mutant IDH1 could not block G-CSF induced Gr.1 expression (59.6% with doxycycline vs 59.2% without doxycycline, respectively; p=0.035). In accordance to these analyses of surface markers, we found that reduction of expression levels of CEBPβ induced by tetracycline-inducible ectopic expression of mutant IDH1 was only observed in 32D-cl3 cells with mutant ASXL1. Furthermore, we found that growth rate of 32D-cl3 cells with mutant ASXL1 and mutant IDH1 was significantly higher than those with mutant ASXL1 alone. These findings suggested that mutant IDH1 can exert its inhibitory effect of normal myeloid differentiation only when mutant ASXL1 is present. Herein, we hypothesized that mutant ASXL1 and mutant IDH1 had unknown cooperative mechanisms to suppress normal differentiation and promote leukemogenesis, and performed transcriptome analysis of the 32D-cl3 cells treated with G-CSF. Tendency toward decreased expression of genes associated with myeloid differentiation in the presence of IDH1 was only observed in cells with mutant ASXL1. Further, it was revealed that specifically dysregulated genes such as HDAC6 or KMT2A in leukemic stem cells were enriched in 32D-cl3 cells with mutant ASXL1, which suggested that mutant IDH1 could induce leukemogenesis only in the presence of ASXL1. In our study, we found that the presence of IDH1 R132H mutation in 32D-cl3 cells had an inhibitory effect on normal myeloid differentiation only in the presence of ASXL1 mutation. Transcriptome analysis was performed to explore genes dysregulated by cooperation of mutant ASXL1 and mutant IDH1. We demonstrated that there might be cooperative mechanisms between ASXL1 and IDH1 to induce differentiation and promote leukemogenesis. Our study would not only provide a new role for IDH1 mutation in ASXL1 mutated AML, but also identify a new therapeutic target for ASXL1-mutatated AML.

C/EBPα-p30 Confers AML Cell Susceptibility to the Terminal Unfolded Protein Response and Resistance to Venetoclax By Activating DDIT3 Transcription

Introduction: CCAAT-enhancer-binding protein α (C/EBPα) is one of the most essential lineage-specific transcription factors involved in myeloid differentiation and includes two isoforms of protein: full-length C/EBPα-p42 and truncated C/EBPα-p30. Acute myeloid leukemia (AML) with CEBPA biallelic (CEBPAbi) mutation or single mutation in the bZIP region is associated with a favorable prognosis, but the mechanisms are still unclear. The unfolded protein response (UPR) is a conserved adaptive signal transduction pathway triggered by endoplasmic reticulum (ER) stress. UPR first strives to restore protein homeostasis and maintain cell survival through a pro-survival arm called adaptive UPR. However, in the setting of irremediable ER stress, the UPR transforms into pro-apoptotic terminal UPR signaling that promotes cell apoptosis. DNA damage-inducible transcript 3 (DDIT3) is a key transcription factor responsible for terminal UPR under severe ER stress. Here, we hypothesize that C/EBPα two isoforms regulate DDIT3 transcription, resulting in easier elimination of AML cells with CEBPA mutations and dysregulation by terminal UPR. Methods: Human AML cell lines and mouse 32Dcl3 were transduced with recombinant lentiviruses to knockdown CEBPA expression or over-express of C/EBPα two isoforms. RT-qPCR was used to detected the mRNA levels and western immunoblotting was used to determine the protein levels. The cell apoptosis rates were determined as the percentage of Annexin V-positive cells by flow cytometry. We performed CFU assays to confirm the declined granulocytic differentiation of 32Dcl3 cells with Cebpa knockdown. To identify potential C/EBPα-binding DNA sequences in the genome, we performed ChIP-seq. Luciferase reporter analysis was used to confirm the transcriptional regulation ability of C/EBPα two isoforms and independent ChIP-qPCR of candidate sequences were performed to further verify the transcription factor binding sites of C/EBPα. Finally, we generated a xenograft model with NCG mice to determine the potential application value of our findings. Results: Firstly, we found a negative correlation between CEBPA expression and DDIT3 levels in AML cell lines and primary de novo AML patients. After knockdown of CEBPA in THP-1 cells, which has high endogenous C/EBPα expression, DDIT3 expression was upregulated, and an increased apoptotic rate of AML cells was induced by ER stress. Cebpa knockdown also induced easier elimination of mouse myeloid progenitor cells 32Dcl3 due to upregulation of Ddit3, so as to avoid leukemogenesis since their differentiation was blocked. After the overexpression of C/EBPα-p42 and C/EBPα-p30 in NB4 cells, which have relatively low levels of endogenous C/EBPα isoforms, we found that C/EBPα-p30 activated DDIT3 expression to increase the sensitivity of AML cells to tunicamycin. However, C/EBPα-p42 had the opposite effect to C/EBPα-p30. Through luciferase reporter analysis, we confirmed that C/EBPα-p30 and C/EBPα-p42 directly regulate the transcription of DDIT3 in a contrasting manner, indicating that the two isoforms maintain protein homeostasis corporately. ChIP-seq and ChIP-qPCR revealed that C/EBPα-p30 has a special binding site in DDIT3 gene promoter with stronger affinity than C/EBPα-p42 (hg38, Chr12:57520654-57521026). Given that the BCL2 family proteins are major targets of DDIT3, we further observed that a low p42/p30 ratio was related to reduced BCL2 levels-a major target of DDIT3, increased MCL1 levels, and resistance of AML cells to venetocalx. Finally, in vitro and in vivo we found that tunicamycin and sorafenib could synergistically reverse venetoclax resistance in AML cells by activating the terminal UPR and inhibiting MCL1. Conclusions: Collectively, our results identified a critical regulation of C/EBPα two isoforms on the transcription of DDIT3 in AML cells as well as normal myeloid progenitor cells and confirmed the importance of this regulation in leukemia surveillance and elimination of AML cells. Moreover, we demonstrated that patients with AML having a low C/EBPα p42/p30 ratio and CEBPAbi mutation may not benefit from monotherapy with BCL2 inhibitors. This can be resolved by a combination of ER stress inducers or MCL1 inhibitors, including tunicamycin and sorafenib, which may represent a novel therapeutic strategy for AML patients with CEBPA mutations and dysregulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 823: ABL1 kinase regulates leukemia phenotype and response to therapy

Abstract The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase. Cytoplasmic ABL1 stimulates, whereas nuclear ABL1 inhibits cell growth. Analysis of the CGAP Mitelman database detected loss of ABL1 allele in hematologic malignancies. Among the top genetic aberrations detected in acute myeloid leukemias were t(8;21) generating fusion protein AML1-ETO and NUP98 translocations, e.g., t(1;11) encoding NUP98-PMX1.Expression of AML1-ETO and NUP98-PMX1 in Lin-cKit+ stem cell enriched bone marrow cells from Abl1-/-;Vav-Cre and Abl1+/+;Vav-Cre mice revealed that oncogene-expressing Abl1-/- cells gained tremendous proliferation advantage. Conversely, overexpression of ABL1 and DPH (ABL1 kinase agonist)-mediated activation of ABL1 kinase resulted in reduced proliferation of AML1-ETO and NUP98-PMX1 cells. Since the role of ABL1 in DNA damage response (DDR) is well established, we applied the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi) to identify potential therapeutic vulnerabilities. AML1-ETO;Abl1-/- cells were more sensitive to all three inhibitors when compared to AML1-ETO;Abl1+/+ cells. Remarkably, NUP98-PMX1;Abl1-/- cells were exceptionally sensitive to ATRi, but not to ATMi and DNA-PKi when compared to NUP98-PMX1;Abl+/+ cells. ABL1 did not affect the sensitivity of transformed cells to DNA damaging agent doxorubicin. To pinpoint mechanisms collaborating with Abl1 deletion in transformation of hematopoietic cells, Abl1 was knockout in 32Dcl3 cells by CRISPR/Cas9 to obtain 32Dcl3-Abl1ko cells. 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells were challenged to induce transformation by starving them from growth factors (IL3). Only 32Dcl3-Abl1ko cell populations generated growth factor-independent cells. This indicates that the absence of Abl1 may facilitate malignant transformation of hematopoietic cells. RNA-seq was performed to compare gene expression in 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells in the presence or absence of IL-3. Pathway analysis of FPKM reads (top 30 significant canonical pathways) and Venn diagram analysis followed by KEGG pathway analysis (291 genes uniquely expressed in growth factor-independent 32Dcl3-Abl1ko cells) implicated PI3K-AKT pathway signaling including mTOR and sirtuins (SITR) in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib, mTOR inhibitor rapamycin and SIRT2 inhibitor thiomyristoyl exerted selective activity against Lin-cKit+ AML1-ETO;Abl1-/- and/or NUP98-PMX1;Abl1-/- cells when compared to Abl1+/+ counterparts. Moreover, inhibition of ABL1 kinase by imatinib in Lin-cKit+ AML1-ETO;Abl1+/+ and/or NUP98-PMX1;Abl+/+ cells induced the sensitivity to PI3K-ATK pathway inhibitors. In conclusion, we showed that normal ABL1 kinase plays a tumor suppressor role and is a potential therapeutic target in hematological malignancies induced by AML1-ETO and NUP98-PMX1. The latter statement is being currently tested in animal models. Citation Format: Konstantin Golovine, Zhaorui Lian, Kumaraswamy Naidu Chitrala, Gleb Ablakov, Margaret Nieborowska-Skorska, Jian Huang, Tomasz Skorski. ABL1 kinase regulates leukemia phenotype and response to therapy [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 823.

MS4A3 Promotes Differentiation in Chronic Myeloid Leukemia By Enhancing Common β Chain Cytokine Receptor Endocytosis

MS4A3 Promotes Differentiation in Chronic Myeloid Leukemia By Enhancing Common β Chain Cytokine Receptor Endocytosis

GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

Acute myeloid leukemia (AML) with high expression of a transcriptional factor, Ecotropic viral integration site 1 (EVI1), is associated with extremely poor prognosis. EVI1 is, however, also essential for maintaining normal hematopoietic stem cells (HSCs), rendering it potentially difficult to target this molecule. To overcome this therapeutic difficulty, it is important to comprehensively elucidate differentially regulated downstream targets between normal and leukemia cells. In this study, we searched downstream targets of EVI1 in normal hematopoiesis by combining a chromatin immunoprecipitation sequence (ChIP-seq) and RNA-sequence (RNA-seq) analysis using a mouse hematopoietic cell line 32D-cl3 with high EVI1 expression. We deleted Evi1 using CRISPR/Cas9 in 32D-cl3 cells. Evi1 knock-out (KO) 32D-cl3 cells showed comparable cell growth with parental cells in the presence of IL-3, which enables them to proliferate permanently without differentiation. When they are allowed to differentiate by adding G-CSF, the number of KO cells decreased sharply at day 5-6, compared with parental 32D-cl3 cells. Along with the decreased cell number, KO cells also demonstrated higher positive rate of Gr-1 at day 7, a typical marker of differentiation into granulocytes, indicating accelerated differentiation of KO cells. These results indicated that EVI1 is required to maintain undifferentiated status of 32D-cl3 cells in a differentiation-permissive conditions, which can model normal hematopoiesis. We knocked in 3×FLAG tag at the 3' end of the Evi1 gene to perform ChIP-seq using anti-FLAG antibody. By using these knock-in cells, ChIP-seq was performed on day 0 and day 3 of G-CSF treatment, when they had started to differentiate with still maintained EVI1 expression. The peaks observed in undifferentiated day 0 sample were considered to contain a group of genes involved in undifferentiated hematopoietic cells in cooperation with EVI1. Genes associated HDAC class I, RAC1 signaling were enriched in these genes. To investigate the functional implications of the result of ChIP-seq, RNA-seq data using two clones of KO cells and parental cells were combined. We found that 152 genes were significantly up-regulated, and 155 genes were down-regulated in the KO cells, with false discovery rate less than 0.05. Twenty-four genes were identified by extracting common genes between ChIP-seq and RNA-seq; namely, genes which had day 0-specific peaks in ChIP-seq, and whose expression were decreased in the KO cells. In order to further examine the physiological implications of 24 genes in vivo, we referred to the results of RNA-seq using murine bone marrow transplantation model, where murine hematopoietic progenitor cells retrovirally transduced with Evi1 were transplanted into irradiated syngeneic mice, finally leading to AML after a long latency. Samples obtained early after post transplantation and those after AML onset were compared to those of normal hematopoietic progenitor cells. Among the above 24 genes, the expression of 5 genes was increased early after transplantation and decreased after the onset of AML, that is, these genes were up-regulated by EVI1 but don't seem to be involved in AML maintenance. We functionally validated the role of these genes in 32D-cl3 cells. Of the above, CRISPR/Cas9-mediated knock-out of Gfi1(Growth Factor Independent 1 Transcriptional Repressor) and Mfsd2b (Major facilitator superfamily domain containing 2B) in 32D-cl3 cells led to high Gr-1 positivity at day 7 like Evi1-KO cells, suggesting that these genes are involved in the functions of EVI1 in the normal hematopoiesis. The mRNA expression of these genes was compared in LSK (Lineage- Sca1+ c-kit+) cells from the bone marrow of Evi1 conditional knockout (cKO) mice and control mice. The expression of Gfi1 and Mfsd2b was decreased in LSK cells from Evi1 cKO mice. Furthermore, retroviral expression of Gfi1 in LSK cells restored the reduced colony-forming ability of Evi1 cKO cells. These results collectively suggest that GFI1 is regulated by EVI1 and is involved in the function of EVI1 regulating the stemness of hematopoietic stem and progenitor cells in normal hematopoiesis. These findings provide us with the novel insights on EVI1-mediated HSC maintenance as well as on the therapeutic strategy that specifically targets leukemia-specific EVI1 effectors while preserving normal hematopoiesis. Disclosures Kurokawa: Shire Plc: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Teijin: Research Funding; Takeda: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau.

HMGA1 Is Upregulated in Myelodysplastic Syndromes with Mutation in Pre-mRNA Splicing Genes and Inhibits Leukemia Cell Differentiation

In myelodysplastic syndrome (MDS), genes associated with pre-mRNA splicing such as SRSF2 (Serine and Arginine Rich Splicing Factor 2) and U2AF1 (U2 small nuclear RNA auxiliary factor 1) are frequently mutated. While several reports demonstrated that MDS patients with these mutations had worse prognosis, few treatments specific to these mutations are available, partly due to the dearth of material on functions of these mutated genes. To assess the role of pre-mRNA splicing gene mutations in the pathogenesis of MDS, we performed RNA-seq analysis using CD34-postitive fractions of bone marrow samples of MDS patients. Patients with mutations in either SRSF2 or U2AF1 tended to have shorter 1-year leukemia free survival compared to patients without these mutations (66.7% vs 73.7%, respectively; p=0.067). Gene set enrichment analysis revealed that MYC target genes were enriched in patients with mutations in either SRSF2 or U2AF1 compared to patients (n=4) without these mutations (n=3). Among them, we found that HMGA1 (High Mobility Group A1) was distinctly upregulated in these patients. Expression levels of HMGA1 are known to be higher in hematopoietic stem cells. HMGA1 is involved in various cellular processes such as transcriptional regulation, DNA repair, cell differentiation and regulated cell death. We confirmed that expression levels of HMGA1 in murine bone marrow cells were higher in Lineage marker-negative, Sca1-positive, and c-Kit-positive (LSK) fractions than other more differentiated fractions. To determine whether upregulation of HMGA1 contributed to pathogenesis of MDS, we retrovirally transduced HMGA1 to murine hematopoietic progenitor cell line 32D-cl3. We found that proportions of Gr-1 positive cells after treatment with G-CSF were lower in HMGA1-transduced 32D-cl3 cells. Further, expression levels of myeloid-associated genes were also suppressed in these 32D-cl3 cells. These results suggested that 32D-cl3 with extrinsic expression of HMGA1 were more resistant to G-CSF induced myeloid differentiation. Similarly, primary murine hematopoietic progenitor cells retrovirally transduced with HMGA1 showed increased colony-forming capacities. Considering that differentiation block plays a major role in the onset of MDS, these results supported our hypothesis that upregulation of HMGA1 would contribute to pathogenesis of MDS through blocking normal myeloid differentiation. To investigate whether inhibition of HMGA1 could affect the pathogenesis of myeloid malignancies, we silenced expression of HMGA1 via short-hairpin RNA (shRNA) in various cell lines of acute myeloid leukemia (AML). Human acute monocytic leukemia cell line THP-1 became more easily induced to differentiate upon phorbol12-myristate13-acetate treatment when HMGA1 was silenced. Similarly, we silenced expression of HMGA1 in HL60, which is a human acute promyelocytic leukemia (APL) cell line. We treated HL60 cells with all-trance retinoic acid (ATRA) to induce myeloid differentiation. As expected, we found that expression levels of CEBP-β and Gr-1 in ATRA-treated HL60 cells were significantly higher when HMGA1 was silenced, which meant that HL60 cells became more sensitive to ATRA-induced myeloid differentiation by inhibition of HMGA1. Considering that differentiation induction therapy has improved clinical outcome not only in APL but also in AML, these results suggested that HMGA1 would be a potential therapeutic target for myeloid malignancies. In summary, our experiments demonstrated that expression levels of HMGA1 were higher in patients with splicing mutations. Considering its inhibitory effects on differentiation demonstrated by our experiments, upregulation of HMGA1 could be involved in the pathogenesis of MDS. Further, therapeutic intervention for HMGA1 had a potential to improve clinical outcome of myeloid malignancies. Disclosures Kurokawa: Nippon Shinyaku: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Teijin: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bioverativ Japan: Consultancy; Shire Plc: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; Chugai: Consultancy, Research Funding, Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau.

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinases Exerts Synergistic Effect Against Chronic Myeloid Leukemia Cells

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of BCR-ABL1 tyrosine kinase - positive chronic myeloid leukemia in chronic phase (CML-CP). However, it is unlikely that TKIs will "cure" the disease in majority of patients because CML-CP cells are elusive targets even for the most advanced therapies employing second and third generation of TKIs. Therefore, new treatment modalities are necessary to improve therapeutic outcomes. We showed before that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. The aim of the study was to test whether simultaneous inhibition of signaling pathways activated by BCR-ABL1 and PAK kinases may improve the treatment of CML. Special attention was focused on PAK1 and PAK2, which are expressed in hematopoietic cells and can play an important role in the promotion of CML cells proliferation and survival. PAK kinases were targeted by small molecule inhibitor IPA-3 (inhibitor of PAK1) and shRNA construct for PAK2, BCR-ABL1 kinase was inhibited by imatinib. The studies were carried out using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed CML-CP patients and healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) BCR-ABL1-transformed 32Dcl3 cell line cells. We show here that inhibition of PAK1 or/and PAK2 kinases activity enhanced the effect of IM against CML cells without affecting normal counterparts. We observed that the combined use of IM with IPA-3 increased growth inhibition and apoptosis of leukemia cells. To evaluate the type of drugs interaction median effect analysis method was used. The studies revealed that the type and strength of drug interaction depend on drug concentration. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Altogether, we postulate that BCR-ABL1 kinase inhibitor should be combined with PAK1/2 inhibitor to facilitate eradication of CML cells. Disclosures No relevant conflicts of interest to declare.

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinase Exerts Synergistic Effect against Chronic Myeloid Leukemia Cells.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in the chronic phase (CML-CP). However, it is unlikely that they can completely “cure” the disease. This might be because some subpopulations of CML-CP cells such as stem and progenitor cells are resistant to chemotherapy, even to the new generation of TKIs. Therefore, it is important to look for new methods of treatment to improve therapeutic outcomes. Previously, we have shown that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. In this study, we aimed to determine if simultaneous inhibition of BCR-ABL1 oncogenic tyrosine kinase and PAK1/2 serine/threonine kinase exert better anti-CML effect than that of individual treatments. PAK1 was inhibited by small-molecule inhibitor IPA-3 (p21-activated kinase inhibitor III), PAK2 was downregulated by specific short hairpin RNA (shRNA), and BCR-ABL1 tyrosine kinase was inhibited by imatinib (IM). The studies were conducted by using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed patients with CML-CP and from healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) from BCR-ABL1-transformed 32Dcl3 cell line. Herein, we show that inhibition of the activity of PAK1 and/or PAK2 enhanced the effect of IM against CML cells without affecting the normal cells. We observed that the combined use of IM with IPA-3 increased the inhibition of growth and apoptosis of leukemia cells. To evaluate the type of interaction between the two drugs, we performed median effect analysis. According to our results, the type and strength of drug interaction depend on the concentration of the drugs tested. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Based on our results, we hypothesize that IM, a BCR-ABL1 tyrosine kinase inhibitor, combined with a PAK1/2 inhibitor facilitates eradication of CML-CP cells.

Abstract 4643: Mutant ASXL1 collaborates with HHEX to promote myeloid leukemogenesis

Abstract An epigenetic modulator Additional sex combs-like 1 (ASXL1) is recurrently mutated in myeloid neoplasms and its mutations are associated with poor prognosis. Recently, we generated mutant Asxl1 conditional knock-in (Asxl1-MT KI) mice mimicking human ASXL1 E635RfsX15 mutation, one of the most common mutations in myeloid neoplasms (Nagase et al. JEM 2018). Retrovirus-mediated insertional mutagenesis study exhibited susceptibility of Asxl1-MT KI bone marrow cells to myeloid leukemia, and we identified Hematopoietically expressed homeobox (Hhex) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between the mutant ASXL1 and HHEX in myeloid leukemogenesis. We first performed colony-forming assay and found that forced expression of HHEX enhanced colony replating activity and blocked myeloid differentiation in bone marrow hematopoietic stem progenitor cells (HSPCs) derived from ASXL1-MT KI mice, while it showed only modest effect in normal HSPCs. The synergistic effect between the mutant ASXL1 and HHEX in blocking myeloid differentiation was also observed in human HL-60 cells. We next evaluated the role of endogenous Hhex in the mutant ASXL1-expressing cells. Depletion of endogenous Hhex using CRISPR-Cas9 system ameliorated mutant ASXL1-induced differentiation block in 32Dcl3 cells. Depletion of endogenous Hhex in murine mutant ASXL1-expressing leukemia cells [cSAM cells: cells with combined expression of SETBP1 and ASXL1 mutations (Inoue et al. Leukemia 2015), cRAM cells: cells with combined expression of RUNX1 and ASXL1 mutations (Nagase et al. JEM 2018)] also promoted differentiation and increased apoptosis. Furthermore, Hhex deletion profoundly attenuated the colonogenicity of cSAM and cRAM cells and leukemogenicity of cSAM cells. We then investigated target genes of the mutant ASXL1 and HHEX in myeloid neoplasms using public database and our previous RNA-Seq data. Among the potential target genes of the mutant ASXL1 and HHEX, we found that Myb, Etv5 and Oraov1 genes were upregulated by the mutant ASXL1 and HHEX in murine HSPCs. Conversely, Hhex depletion resulted in downregulation of these genes both in cSAM and cRAM leukemic cells. In addition, depletion of Myb, Etv5 or Oraov1 genes significantly abrogated the colonogenicity of cSAM cells. These data suggest that mutant ASXL1 and HHEX cooperatively induce myeloid leukemogenesis via dysregulating Myb, Etv5 and Oraov1. Citation Format: Shuhei Asada, Reina Takeda, Daichi Inoue, Susumu Goyama, Toshio Kitamura. Mutant ASXL1 collaborates with HHEX to promote myeloid leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4643.

HoxA9 binds and represses the Cebpa +8 kb enhancer.

C/EBPα plays a key role in specifying myeloid lineage development. HoxA9 is expressed in myeloid progenitors, with its level diminishing during myeloid maturation, and HOXA9 is over-expressed in a majority of acute myeloid leukemia cases, including those expressing NUP98-HOXD13. The objective of this study was to determine whether HoxA9 directly represses Cebpa gene expression. We find 4-fold increased HoxA9 and 5-fold reduced Cebpa in marrow common myeloid and LSK progenitors from Vav-NUP98-HOXD13 transgenic mice. Conversely, HoxA9 decreases 5-fold while Cebpa increases during granulocytic differentiation of 32Dcl3 myeloid cells. Activation of exogenous HoxA9-ER in 32Dcl3 cells reduces Cebpa mRNA even in the presence of cycloheximide, suggesting direct repression. Cebpa transcription in murine myeloid cells is regulated by a hematopoietic-specific +37 kb enhancer and by a more widely active +8 kb enhancer. ChIP-Seq analysis of primary myeloid progenitor cells expressing exogenous HoxA9 or HoxA9-ER demonstrates that HoxA9 localizes to both the +8 kb and +37 kb Cebpa enhancers. Gel shift analysis demonstrates HoxA9 binding to three consensus sites in the +8 kb enhancer, but no affinity for the single near-consensus site present in the +37 kb enhancer. Activity of a Cebpa +8 kb enhancer/promoter-luciferase reporter in 32Dcl3 or MOLM14 myeloid cells is increased ~2-fold by mutation of its three HOXA9-binding sites, suggesting that endogenous HoxA9 represses +8 kb Cebpa enhancer activity. In contrast, mutation of five C/EBPα-binding sites in the +8 kb enhancer reduces activity 3-fold. Finally, expression of a +37 kb enhancer/promoter-hCD4 transgene reporter is reduced ~2-fold in marrow common myeloid progenitors when the Vav-NUP98-HOXD13 transgene is introduced. Overall, these data support the conclusion that HoxA9 represses Cebpa expression, at least in part via inhibition of its +8 kb enhancer, potentially allowing normal myeloid progenitors to maintain immaturity and contributing to the pathogenesis of acute myeloid leukemia associated with increased HOXA9.

Molecular mechanisms for enhancement of stromal cell-derived factor 1–induced chemotaxis by platelet endothelial cell adhesion molecule 1 (PECAM-1)

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration. Interestingly, several PECAM-1-deficient hematopoietic cells exhibit impaired chemotactic responses to stromal cell-derived factor 1 (SDF-1), a chemokine essential for B lymphopoiesis and bone marrow myelopoiesis. However, whether PECAM-1 is involved in SDF-1-regulated chemotaxis is unknown. We report here that SDF-1 induces tyrosine phosphorylation of PECAM-1 at its immunoreceptor tyrosine-based inhibition motifs in several hematopoietic cell lines via the Src family kinase Lyn, Bruton's tyrosine kinase, and JAK2 and that inhibition of these kinases reduced chemotaxis. Overexpression and knockdown of PECAM-1 enhanced and down-regulated, respectively, SDF-1-induced Gαi-dependent activation of the PI3K/Akt/mTORC1 pathway and small GTPase Rap1 in hematopoietic 32Dcl3 cells, and these changes in activation correlated with chemotaxis. Furthermore, pharmacological or genetic inhibition of the PI3K/Akt/mTORC1 pathway or Rap1, respectively, revealed that these pathways are independently activated and required for SDF-1-induced chemotaxis. When coexpressed in 293T cells, PECAM-1 physically associated with the SDF-1 receptor CXCR4. Moreover, PECAM-1 overexpression and knockdown reduced and enhanced SDF-1-induced endocytosis of CXCR4, respectively. Furthermore, when expressed in 32Dcl3 cells, an endocytosis-defective CXCR4 mutant, CXCR4-S324A/S325A, could activate the PI3K/Akt/mTORC1 pathway as well as Rap1 and induce chemotaxis in a manner similar to PECAM-1 overexpression. These findings suggest that PECAM-1 enhances SDF-1-induced chemotaxis by augmenting and prolonging activation of the PI3K/Akt/mTORC1 pathway and Rap1 and that PECAM-1, at least partly, exerts its activity by inhibiting SDF-1-induced internalization of CXCR4.

Pathways Relevant to AML Pathogenesis Targeting the Hematopoietic-Specific Cebpa +37 Kb Enhancer

C/EBPα mediates myeloid differentiation, and its reduced activity is central to myeloid transformation (Friedman Int. J. Hematol. 2015). The murine Cebpa gene contains a 450 bp, +37 kb element that acquires the enhancer-specific H3K4me1 and H3K27Ac histone marks as LT-HSC progress to GMP and directs hCD4 transgene expression to GMP and myeloid CFUs (Guo et al. Blood 2012; Guo et al. J. Leuk. Biol. 2014). Moreover, CRISPR/Cas9 mediated, biallelic replacement of the enhancer with a variant harboring mutations in its seven Ets sites reduces Cebpa RNA >10-fold in 32Dcl3 cells (Cooper et al. PLoS One 2015), and germline or Cre-mediated enhancer deletion leads to marked reduction in Cebpa RNA in marrow LSK, CMP, and GMP, with 3-fold reduction in GMP, CFU-G, and neutrophils and indefinite myeloid colony replating in IL-3, a preleukemic phenotype (Guo et al. PLoS One 2016; Avellino et al. Blood 2016). The enhancer contains four conserved RUNX1 cis elements that bind RUNX1 in gel shift or ChIP assays, Runx1 gene deletion reduces Cebpa RNA 5-fold in Lin- and 2-fold in GMP marrow cells, Runx1 cis element mutation reduces luciferase reporter activity 6-fold in 32Dcl3 myeloid cells (Guo et al. Blood 2012), and in AMLs with t(8;21) RUNX1-ETO interacts with the human CEBPA locus at the homologous +42 kb enhancer (Ptasinska et al. Leukemia 2012). Mutation of the CEBPA enhancer has not been seen in human AML cases, perhaps reflecting preference for upstream pathway alteration to suppress both alleles and additional genes. As a further example of a leukemic alteration affecting enhancer activity, elevated EVI1 is a high-risk feature in AML, and EVI1 binds and represses the +37 kb Cebpa enhancer (Wilson et al. J. Biol. Chem. 2016). We have characterized additional pathways whose modulation in AML may reduce CEBPA enhancer activity. Deletion of the Pu.1 -14 kb enhancer leads to AML in mice, and PU.1 binds the Cebpa enhancer in ChIP and gel shift; we now find that mutation of the one Cebpa enhancer Ets site that binds PU.1 reduces reporter activity 4-fold in 32Dcl3 cells, providing the first functional evidence that PU.1 regulates the enhancer. ~10% of human AMLs harbor mutant C/EBPα proteins, CEBPα binds the enhancer in ChIP and gel shift, and mutation of the two enhancer C/EBP elements reduces reporter activity; we have now used CRISPR/Cas9 to generate a 32Dcl3 cell line with the two enhancer C/EBPsites mutated in one allele and find 60% reduction in Cebpa RNA expression. FLT3/ITD is a constitutively activated mutant form of the receptor tyrosine kinase found in ~30% of AML cases and confers high risk. While FLT3/ITD predominantly provides proliferative signals, it may also contribute to impaired differentiation, as FLT3/ITD inhibition in AML cases leads to a neutrophilic differentiation syndrome. We previously demonstrated that FLT3/ITD reduces Cebpa RNA in 32Dcl3 cells and patient leukemic blasts and that the first generation FLT3/ITD inhibitor lestaurtinib increases Cebpa; we now find that the effect on Cebpa RNA is also reversed in 32Dcl3 cells by the next generation inhibitor crenolanib, that FLT3/ITD reduces Cebpa enhancer H3K4me1 and H3K27Ac histone marks several-fold, and that both of these activating marks are also restored by crenolanib. FLT3/ITD signaling leads to C/EBPα serine phosphorylation to reduce its activity, which may account in part for the inhibitory effect of FLT3/ITD on Cebpa enhancer activity. Finally, NUP98-HOX fusion proteins contribute to a small percent of AML cases. Vav-NUP98/HOXD13 (NHD13) mice develop MDS and can progress to AML; we now find that Cebpa RNA is reduced 5-fold in Vav-NHD13 CMP and LSK, with 5-fold increased HoxA9 RNA, that Vav-NHD13::Cebpa Enh-hCD4 compound heterozygous mice have reduced hCD4 expression in GMP and CMP, implicating a direct effect on enhancer activity, and that expression of NHD13 in 32Dcl3 subclones prevents G-CSF induction of Cebpa, MPO, and morphologic differentiation. These 32Dcl3 lines retain levels of Gcsfr equal to empty vector-transduced cells in IL-3 and manifest increased HoxA9 in response to G-CSF. Moreover, HoxA9 binds the Cebpa enhancer in ChIP using marrow-derived HoxA9/Meis1 myeloid lines. Potentially, NHD13 induces HoxA9, which then binds the Cebpa enhancer directly or via other factors to repress transcription. Identifying mechanisms that repress CEBPA hematopoietic enhancer activity in AML may identify approaches to induce differentiation. DisclosuresNo relevant conflicts of interest to declare.

Acetylation of C/EBPα inhibits its granulopoietic function.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation.

Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis

Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the -14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow.

In Vivo Deletion of the Cebpa +37 Kb Enhancer Markedly Reduces Cebpa mRNA in Myeloid Progenitors but Not in Non-Hematopoietic Tissues to Impair Granulopoiesis

The C/EBPα transcription factor is a key mediator of normal myeloid differentiation, and its reduced expression or activity is central to myeloid transformation, as recently reviewed (Friedman, Int. J. Hematol. 2015). The murine Cebpa gene contains a conserved, 450 bp, +37 kb regulatory element that displays the enhancer specific H3K4me1 and the activating H3K27Ac histone modifications at increasing levels as LT-HSC progress to ST-HSC, CMP, and GMP, increases promoter activity 6-fold when introduced into 32Dcl3 myeloid cells linked to a luciferase reporter, directs murine hCD4 transgene expression preferentially to GMP, myeloid CFUs, and functional LT-HSC, and is bound and activated by RUNX1, PU.1, SCL, GATA-2, C/EBPα, and c-Myb (Guo et al, Blood 2012; Guo et al, J. Leuk. Biol., 2014; Cooper et al, PLoS One 2015). In addition, CRISPR/Cas9 mediated enhancer mutation markedly reduces Cebpa transcription in 32Dcl3 cells (Cooper et al, 2015), and in AML samples harboring t(8;21) the RUNX1-ETO oncoprotein interacts with the human CEBPA locus specifically at the homologous +42 kb enhancer. We have now characterized mice harboring floxed Cebpa +37 kb enhancer alleles. Utilizing a B6 BAC encompassing the Cebpa locus, we assembled a targeting vector containing a 5.5 kb 5' homology arm, the +37 kb enhancer flanked by lox P sites, a PGK-Neo cassette flanked by frt sites, and a 0.9 kb 3' homology arm. After introduction into a B6 mESC line, several G418-resistant subclones were isolated, and homologous, heterozygous targeting was confirmed by 3' PCR and 5' and 3' Southern blotting. Two ESC lines were utilized to develop germ line chimeras, and upon breeding with B6 albino mice, one yielded several offspring with germ line transmission of the targeted allele, which were then bred to homozygosity. In an initial set of experiments, marrow from wild-type (WT) and Enh(f/f) mice exposed to 5-FU was transduced with pBabePuro or pBabePuro-Cre followed by puromycin selection and in vitro analysis. Cebpa mRNA was reduced 10-fold in the lineage-negative subset upon Enh(f/f) marrow transduction with Cre. CFU-G were reduced 8-fold, whereas CFU-M and CFU-GM were unaffected, and enhancer deletion enabled serial replating of myeloid CFUs for >16 generations, though these remained dependent on IL-3. Morphologically, enhancer deletion led to depletion of mature granulocytes and accumulation of CD11b- Gr-1- blasts after four days of culture in IL-3, IL-6, and SCF. We next generated Enh(f/f);Mx1-Cre mice and subjected these and Enh(f/f) littermates to pIpC injections, followed by analysis four weeks after the last injection. Cebpa mRNA was reduced 12-fold in GMP, 13-fold in CMP, and >20-fold in the LSK marrow subset. Peripheral blood and marrow neutrophils were reduced 3- to 8-fold, whereas monocytes, RBCs, platelets, and B or T lymphocytes were unaffected. GMP as a percentage of Lin- Sca-1- c-kit+ progenitors were reduced 3-fold, CMP were increased 1.5-fold, and MEP and CLP were unaffected. CFU-G were reduced 3-fold, CFU-M were increased 2-fold, and BFU-E were increased 4-fold. LSK as a percentage of Lin- cells were increased 3-fold, and MPP and ST-HSC as a percentage of LSK were unaffected, whereas LSK/SLAM were reduced 10-fold, and enumeration of functional LT-HSC by competitive transplantation is in progress. We also generated Enh(f/f);Vav-Cre mice; these demonstrated similarly impaired granulopoiesis, but were obtained at only 12% of the Mendelian ratio, suggestive of embryonic lethality as early death was not observed. In addition, we used FLPo-Cre to delete the Neo cassette; homozygous deletion did not alter myeloid CFU numbers or FACS-defined progenitors. Finally, we utilized CMV-Cre to develop germ line enhancer-deleted mice. As with Vav-Cre, homozygous offspring were obtained far below the predicted numbers. Nevertheless, these expressed Cebpa mRNA at levels equivalent to WT controls in liver, lung, adipose, kidney, small intestine, skeletal muscle, and cardiac tissues, and these organs displayed normal cellular architecture upon hematoxylin-eosin staining. In summary, conditional deletion of the +37 kb Cebpa enhancer in adult mice demonstrates that it acts as a critical, hematopoietic specific enhancer of Cebpa gene expression and that marked Cebpa mRNA down-modulation upon enhancer deletion impairs granulopoiesis and enables serial, cytokine-dependent myeloid CFU replating, a pre-leukemic phenotype. DisclosuresNo relevant conflicts of interest to declare.

The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors.

The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation.

The Tumor Suppressors, MS4A3 and G0S2, Are Downregulated in CML Cells with BCR-ABL1 Kinase-Independent Resistance

Abstract Background: The treatment and survival of chronic myeloid leukemia (CML) patients has greatly improved after the discovery of imatinib; however, disease persistence and drug resistance remain as clinical problems. McWeeney et al. (Blood 2010;115:315-325) identified a gene expression signature predictive of primary cytogenetic resistance to imatinib in treatment-naïve CML chronic phase (CML-CP) patients lacking BCR-ABL1 kinase domain mutations. Comparison of this gene classifier with other studies revealed extensive overlap of resistance genes with genes associated with CML blastic transformation, suggesting that CML-CP patients destined to fail imatinib may exhibit a gene profile reminiscent of advanced CML. Based on rank predictive score from the microarray, the top transcripts found to be dysregulated in newly diagnosed patients who subsequently emerged as imatinib non-responders were: PLCXD2, EGF16, GAS2, RXFP1, ITGA2, MS4A3, FCN1, EMCN, EMCN, CLIP4, ZNF44 and G0S2. Among these, MS4A3 and G0S2 were differentially downregulated in non-responders compared to responders. Conversely, high levels of MS4A3 (p=0.059) and G0S2 (p=0.036) correlated with higher likelihood of major cytogenetic response and longer overall survival. MS4A3 (HTM4) is a hematopoietic cell cycle regulator that inhibits G1/S phase cell cycle transition, whereas G0S2 is proapoptotic mitochondrial protein that interacts with and antagonizes BCL-2. In this study, we investigated the potential role of MS4A3 and G0S2 as tumor suppressors in CML and their influence on TKI resistance and blastic transformation. MS4A3 and CML: Expression of p210BCR-ABL1 in 32Dcl3 or Mo7e myeloid progenitor cells resulted in an 80% reduction of MS4A3 mRNA relative to parental cells by qRT-PCR analysis. Imatinib treatment slightly restored MS4A3 levels in 32D-p210 or Mo7e-p210 cells, but did not return levels to those of normal controls growing with cytokine support. Consistent with a role for MS4A3 in CML blastic transformation, qRT-PCR revealed low levels of MS4A3 in cell line models of blastic phase CML (CML-BP), including K562, KYO-1, and KBM, that were unaffected by treatment with imatinib. Furthermore, qRT-PCR confirmed that MS4A3 is downregulated (~20-fold) in CML CD34+ progenitor cells from CML-BP (n=3) compared to CML-CP (n=5) patients and normal controls (n=3), and that these levels were unaffected by imatinib. We then used tetracycline-inducible shRNA directed against MS4A3 (shMS4A3) to knockdown MS4A3 in primary CML CD34+ cells from newly diagnosed CML-CP patients subsequently responding to TKIs. Western blot and qRT-PCR analyses confirmed MS4A3 downregulation upon exposure to doxycyline (0.1 ug/mL). shMS4A3 upregulated colony formation by 37.6% (p&lt;0.001) in the absence of imatinib, 58.8% (p&lt;0.0001) in 1 µM imatinib, and 138.4% (p&lt;0.001) in 2.5 µM imatinib. Furthermore, shMS4A3 significantly reduced imatinib-induced apoptosis of CML-CP samples in the presence but not absence of doxycycline (p&lt;0.02). G0S2 and CML: Consistent with a role for G0S2 in CML blastic transformation, qRT-PCR revealed that G0S2 mRNA is highly downregulated (~24-fold) in CML CD34+ progenitor cells from CML-BP (n=3) compared to CML-CP (n=5) patients and normal controls (n=3). G0S2 is also downregulated in TKI-resistant K562R and AR230R cells compared to parental TKI-sensitive counterparts. K562R and AR230R cells are resistant to all clinically approved TKIs, but lack BCR-ABL1 kinase domain mutations, implicating BCR-ABL1 kinase-independent TKI resistance. Ectopic expression of a Flag-tagged G0S2 (G0S2-Flag) significantly reduced colony formation in both parental K562 and AR230 cells, but had an even greater effect in TKI-resistant K562R and AR230R cells in the presence of imatinib. G0S2-Flag also impaired colony formation of CML-CP CD34+cells in both the presence (p&lt;0.03) and absence (p&lt;0.01) of imatinib (1 µM). Consistent with a proapoptotic role for G0S2, G0S2-Flag increased Annexin V positivity in all cell lines and patient samples tested. Conclusions:These findings suggest a role for loss of MS4A3 or G0S2 tumor suppressor function in both TKI resistance in the absence of explanatory BCR-ABL1 kinase domain mutations and in CML blastic transformation. Studies to test the effects of restored MS4A3 or G0S2 expression in CML-BP and TKI-resistant patient samples are currently underway. Disclosures Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.