Development Of AML Research Articles

N6-methyladenosine-modified SENP1, identified by IGF2BP3, is a novel molecular marker in acute myeloid leukemia and aggravates progression by activating AKT signal via de-SUMOylating HDAC2

BackgroundElevated evidence suggests that the SENPs family plays an important role in tumor progression. However, the role of SENPs in AML remains unclear.MethodsWe evaluated the expression pattern of SENP1 based on RNA sequencing data obtained from OHSU, TCGA, TARGET, and MILE datasets. Clinical samples were used to verify the expression of SENP1 in the AML cells. Lentiviral vectors shRNA and sgRNA were used to intervene in SENP1 expression in AML cells, and the effects of SENP1 on AML proliferation and anti-apoptosis were detected using in vitro and in vivo models. Chip-qPCR, MERIP-qPCR, CO-IP, RNA pulldown, and dual-luciferase reporter gene assays were used to explore the regulatory mechanisms of SNEP1 in AML.ResultsSENP1 was significantly upregulated in high-risk AML patients and closely related to poor prognosis. The AKT/mTOR signaling pathway is a key downstream pathway that mediates SENP1's regulation of AML proliferation and anti-apoptosis. Mechanistically, the CO-IP assay revealed binding between SENP1 and HDAC2. SUMO and Chip-qPCR assays suggested that SENP1 can desumoylate HDAC2, which enhances EGFR transcription and activates the AKT pathway. In addition, we found that IGF2BP3 expression was upregulated in high-risk AML patients and was positively correlated with SENP1 expression. MERIP-qPCR and RIP-qPCR showed that IGF2BP3 binds SENP1 3-UTR in an m6A manner, enhances SENP1 expression, and promotes AKT pathway conduction.ConclusionsOur findings reveal a distinct mechanism of SENP1-mediated HDAC2-AKT activation and establish the critical role of the IGF2BP3/SENP1signaling axis in AML development.

ЗРОСТАННЯ РОЛІ РЕГТЕХ ЯК СКЛАДОВОЇ БІЗНЕС МОДЕЛІ ФІНАНСОВОЇ УСТАНОВИ У ПЕРІОД ЦИФРОВИХ ТРАНСФОРМАЦІЙ

The article analyzes the reasons for the emergence of Regtech, in particular, the complexity and variability of legislation, the growth of regulatory costs of both financial institutions and regulators, the need for new technologies that would optimize operations to comply with regulatory requirements. Objectives, tools and main characteristics of RegTech analyzed. The division of RegTech technological processes into customer onboarding, constant monitoring, risk identification, and comprehensive reporting is defined. The key features of RegTech are analyzed, namely blockchain, data standardization, high costs, high compliance risks, risk management, importance. RegTech has the potential for a wide range of applications from financial regulation to monitoring corporations for compliance with environmental legislation. Therefore, RegTech gradually outgrows the boundaries of the FinTech sector and becomes a relatively independent direction.The driving forces of the "explosive" growth of the RegTech market are identified, which include the aggravation of the need for post-crisis regulatory changes and the increase in data flows from controlled organizations; growing complexity of regulatory requirements; minimization of growing costs for ensuring compliance with regulatory requirements and standards; technological progress and rapid implementation of advanced technologies; real-time transaction monitoring, threat analysis and secure identity verification; increasing the effectiveness of supervisory tools in order to support financial stability. Innovative trends in the development of RegTech are analyzed, which include the focus on natural language processing, the development of AML and the metauniverse, the spread of cryptocurrencies, distributed ledger technology, voice recognition technology, and geographic information systems. The growing emphasis on business efficiency is increasing the popularity of RegTech among financial institutions, which allows them to increase productivity by reducing regulatory costs, protecting digital data and meeting regulatory requirements.

Monoclonal Antibodies Targeting LIGHT Block Ltbr Signalling and Eliminate Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a molecular heterogeneous disease that originates from the malignant transformation of a hematogenic stem- or progenitor cells resulting in a leukemia stem cell (LSC). LSCs are a main cause of resistance to therapy and relapse. Despite recent advances in targeted therapy and immunotherapy, prognosis for elderly patients not fit do undergo intensive therapy remains poor. We recently documented that targeting CD70, the ligand of the tumor necrosis factor receptor (TNFR) CD27, by the antibody-dependent cell-mediated cytotoxicity (ADCC)-enhanced monoclonal antibody (mAb) cusatuzumab eliminates LSC. We now studied the role of the related TNFR lymphotoxin b receptor (LTbR) with its ligand LIGHT in the pathogenesis of AML in mice and humans and developed a LIGHT targeting mAb to specifically eliminate LSCs. Experimental design We investigated the impact of LIGHT/LTbR signalling in AML development by using the retrovirally-induced MLL-AF9 syngeneic AML mouse model. Light-, Ltbr -deficient and control MLL-AF9 transduced LSCs were injected intravenously into non-irradiated BL/6 recipient mice. AML development was evaluated by analysing MLL-AF9-GFP-positive leukemic cells in blood, spleen, and bone marrow (BM). BM LSC were phenotypically characterized by FACS and functionally by performing colony forming unit (CFU) assays in vitro and by secondary transplantations of LSCs including limited dilution assays (LDA) in vivo. Correlative survival analysis for LIGHT and LTbR expression was performed on a publicly available gene dataset (GSE6891). LIGHT and LTBR protein expression was analysed on primary AML CD34 + leukemic stem and progenitor cells (LSPCs) and control human CD34 + hematopoietic stem cell and progenitor cells (HSCPs). LIGHT/LTbR signalling was blocked by either siRNA treatment or by treatment with novel LIGHT-targeting mAb. The effect of blocking LIGHT/LTbR-signalling was analysed in vitro by CFU assays, RNA-sequencing and xenotransplant experiments in vivo. LIGHT-targeting mAbs were generated by immunization of llamas and phage display selections followed by cloning of FABs into a human backbone (hIgG1). Clones were selected based on their blocking potency of receptor/ligand interaction using competition ELISA and FACS-based cell binding affinity assays. ADCC-enhanced anti-LIGHT mAbs were generated via POTELLIGENT® technology. Anti-LIGHT mAbs that specifically block the interaction of LIGHT/LTBR with deficiency in effector functions (Fc-death) were produced by amino acid substitutions in the CH2 domain of the parental clone. Results LTbR was expressed by HSCs and AML LSCs. In contrast, LIGHT was only expressed on murine and human LSCs but not normal HSCs. The absence of LIGHT or its receptor LTbR on LSCs resulted in significantly prolonged survival and decreased numbers of LSC in BM of AML mice. Light -/- and Ltbr -/- LSCs had a reduced CFU capacity compared to control LSCs. Similarly, inhibition of LIGHT-signalling on LSCs via LTbR-Ig significantly reduced colony formation. Moreover, secondary transplantation of Light -/-LSCs resulted in prolonged survival compared to Light-proficient LSCs. An LDA analysis revealed a 11-fold reduction of the LSC frequency in BM in the absence of LIGHT or LTBR. Similarly, inhibiting LIGHT signalling by siLIGHT RNA in human AML LSPCs resulted in reduced CFU capacity compared to control siRNA treatment. In addition, a GEO analysis revealed that low LIGHT or LTbR mRNA levels correlated with improved overall survival in AML patients. After immunizations of llamas and phage-display selection three humanized anti-LIGHT mAbs with high blocking and high binding efficiency were selected (mAb10F11 and 12C2 specific for human LIGHT and the cross-reactive murine/human 9F6 mAB). Incubation of LSCs with the blocking mAbs significantly reduced CFU capacity. HSPCs from healthy donors remained unaffected by the treatment. A gene expression analysis revealed that blocking LIGHT/LTbR-signalling in LSCs reduced stemness and promoted differentiation. In addition, ADCC-enhanced mAbs activated NK cells to eliminate LIGHT-expressing LSCs. Our findings reveal that LIGHT/LTbR-signalling is crucial for the pathogenesis of AML and especially for the maintenance and expansion of LSCs. We developed and validated novel LIGHT targeting mAbs in vitro and in vivo for further development in clinical trials.

Development of a Fully Immunocompetent Adoptive Transfer In Vivo Model of Npm1 cA;Idh2 R140Q;Flt3 ITD AML

AML is a highly heterogeneous disease, both genetically and phenotypically. This includes both intra- and inter-patient heterogeneity that significantly impacts prognosis and treatment responses. Mouse models are a powerful tool to further our understanding of fundamental disease biology and to advance novel therapies. While human cell line (CDX) and primary patient (PDX) xenografts are reproducible in vivo models, these models are by necessity immunodeficient. Transgenic mouse models provide an opportunity to study disease development driven by human relevant mutations within a fully immunocompetent system. However, drug development studies often require large numbers of mice and predictable time of disease development that is infeasible using primary transgenic animals. We therefore sought to develop a transgenic mouse model representative of human disease that was able to consistently be adoptively transferred into fully immunocompetent recipients. Our laboratory has interest in multiple targeted therapeutics and chose to focus on a model that may have sensitivity to multiple translationally relevant targeted drugs. We crossed the previously established Npm1 cA (Vassiliou et al 2011), Flt3 ITD (Lee et al 2007) and Idh2 R140Q (Shih et al 2017) Cre-inducible alleles to generate a triple mutant AML model ( Npm1 cA;Idh2 R140Q;Flt3 ITD). This model develops an aggressive AML with a median survival of 44 days. Complete blood counts at endpoint demonstrate significantly elevated white cells as well as profound anemia and thrombocytopenia. Npm1 cA;Idh2 R140Q;Flt3 ITD mice also have splenomegaly and infiltration of immature myeloid cells into both lymphoid and non-lymphoid tissues. Flow cytometry analysis of the blood, spleen and bone marrow shows a significant expansion of CD11b+cKit+ cells characteristic of an AML blast phenotype. Given the aggressive nature of this model, we sought to determine whether Npm1 cA;Idh2 R140Q;Flt3 ITD AML cells could be adoptively transferred into either immunodeficient or fully immunocompetent recipients without prior preconditioning. NOD- Prkdc em26Cd52Il2rg em26Cd22/NjuCrl (NCG) and B6.SJL-Ptprca Pepcb/BoyJ (PepBoy) recipients were injected with 1x10 6 bulk splenocytes via the tail vein. Recipient mice quickly develop a lethal AML that faithfully recapitulates the disease observed in the primary transgenic donors. Circulating leukemia cells are detectable from approximately 2 weeks post adoptive transfer (post-AT) and median survival of PepBoy recipients ranged from 17 to 60 days depending on donor (Figure 1). Comprehensive immune profiling using a 36-specificity spectral flow panel showed that in PepBoy recipients, the donor derived cells accurately recapitulate the diversity of the AML immunophenotype of the primary transgenic donor. Notably, cells from male donors do not establish detectable disease in immunocompetent female recipients. This is associated with an increased number of T cells in the female recipients of male cells suggesting immune mediated control of AML development. To assess the response to targeted therapies in a fully immune competent model, we adoptively transferred of our Npm1 cA;Idh2 R140Q;Flt3 ITD to into PepBoy recipients. Mice were randomized to treatment groups based on peripheral blood disease burden at 14 days post AT (0.2-1% of CD45+ cells). Mice received daily oral gavage of either vehicle, 100 mg/kg venetoclax (BCL2 inhibitor), 100 mg/kg enasidenib (IDH2 inhibitor) or 30 mg/kg gilteritinib (FLT3 inhibitor). All three drugs resulted in a significant reduction in circulating tumor burden after 14 days of treatment. Enasidenib did not prolong survival compared to vehicle whereas venetoclax treatment resulted in a significant survival advantage (median 47 days vs 40 in the vehicle). This model was most sensitive to gilteritinib with a median survival of 68 days. (Figure 2). Together these findings demonstrated that the Npm1cA;Idh2 R140Q;Flt3 ITD model is an aggressive model of AML. Adoptive transfer of splenocytes into either immunodeficient NCG or fully immunocompetent PepBoy recipients faithfully recapitulates the disease of primary mice in a reproducible manner. This model therefore provides an in vivo model conducive to drug development studies and provides the opportunity to understand the impact of both AML development and therapeutics on the normal immune system.

Integrative Genome and Transcriptome Sequencing Analysis Indicates Genetic and Epigenetic Dysregulation in DS-AML

Down Syndrome related AML is driven by GATA1 alterations in patients with trisomy 21. Using exome or targeted sequencing, prior studies identified GATA1 alterations in ~80-90% of patients. We performed whole genome sequencing coupled with whole transcriptome sequencing on 207 cases to study the genomic basis of Down Syndrome AML. We detected GATA1 alterations in 96.1% of cases, with additional structural rearrangements in GATA1 in 15% of cases that may have been missed in prior studies. For GATA1, although substitutions and small indels occur nearly uniformly across exon 2, we observed that internal tandem duplications (ITDs) are significantly enriched in the second half of exon 2, suggesting a specific mutational mechanism for these cases. We also detected exon 3 (instead of exon 2) deletion in one case, where the resultant truncated GATA1 protein is different from the commonly recognized sGATA1. Our study further revealed that mutations in genes mediating JAK-STAT signaling (JAK1/JAK2/JAK3), RAS pathway (KRAS/NRAS/NF1), Cohesin complex (STAG2/CTCF/RAD21/SMC3), as well as MBNL1, KANSL1, IRX1, and NFIA. By integrating the genome with transcriptome sequencing data, we discovered that GATA1 exon 2 skipping to be a significant event in DS-AML. Although exon 2 is nearly completely skipped in cases with exon 2 deletion or splice site alterations, significant exon 2 skipping is also observed in cases where the alteration is small and is in the middle of exon 2. Further, exon 2 skipping is also observed in cases wither the alteration is a single base substitution that results in stop codon in the middle of exon 2 (therefore splicing is unlikely affected by these alterations in such cases). Based on this observation, we propose a hybrid genetic-epigenetic model on the development of Down Syndrome AML. In DS-AML, GATA1 exon 2 skipping during transcription/splicing might be a developmentally (epigenetically) regulated event in early fetal development that is destined to be silenced in the postnatal period that results in activation of the canonical long GATA1. This epigenetic silencing of sGATA1 is overridden by secondary genetic events, thus maintaining sGATA1 as the predominant GATA1 protein. Notably, the sGATA1 have variable isoforms as a result of exon 2 or exon 3 loss. In summary, our integrated whole genome and transcriptome approach has resulted in discovery of novel and high-prevalence structural alterations in GATA1 and potentially novel genes involved in pathogenesis of DS-AML. Our integrated analysis provided evidence on the epigenetic/developmental regulation of GATA1 exon 2 skipping via alterative splicing in the context of trisomy 21 that warrants further investigation.

DNMTs-mediated SOCS3 methylation promotes the occurrence and development of AML.

As a tumor suppressor gene, SOCS3 inhibits the growth of tumor cells by regulating JAK/STAT signaling pathway through negative feedback. This study aimed to investigate the biological function and mechanism of SOCS3 methylation mediated by DNMTs in the development of AML. Bone marrow samples were collected from 70 AML patients and 20 healthy volunteers. The expression and methylation status of each gene were detected by RT-qPCR, western blot and MS-PCR, and the growth and apoptosis rate of leukemia cell lines were detected by CCK-8 and flow cytometry. The effects of changes in SOCS3 gene expression and methylation status of AML cell lines were observed by gene transfection and gene knockdown. The methylation rate of SOCS3 in AML initial treatment group was significantly higher than that in the remission group and the normal control group (60% vs. 0%, 0%). The expression of SOCS3 in the SOCS3 methylation group was significantly lower than that in the non-methylated group and control group, while the expression of DNMT1, DNMT3a, p-JAK2, p-STAT3 and p-STAT5 were significantly higher than those in the non-methylated group and control group. Demethylation treatment, SOCS3 transfection and DNMT3a knockdown could up-regulate the expression of SOCS3, which decreased the proliferation and increased the apoptosis of leukemia cell lines. SOCS3 methylation mediated by DNMTs promotes the occurrence and development of AML and can be used as a potential biomarker for the diagnosis and efficacy evaluation of AML.

Trp53-Loss in Hematopoietic Stem Cells Drives the Evolutionary Process of Leukemic Transformation in a Jak2V617F-Driven Myeloproliferative Neoplasms Mouse Model

Myeloproliferative neoplasms (MPNs) are clonal disorders resulting from genetic lesions in hematopoietic stem cells (HSCs). The development of post-MPN AML is treatment resistant and associated with very poor outcomes. This leukemic transformation is due to TP53 inactivation in approximately 50% of cases. Existing mouse models with double mutant Jak2V617F and Trp53 have demonstrated that Trp53-lossalone is sufficient to induce leukemic transformation in Jak2V617F-driven MPN. These models were generated either by crossing Jak2V617F transgenic mice with Trp53R172H knock-in or Trp53 knock-out mice, or by retroviral expression of Jak2V617F in Trp53 knock-out bone morrow (BM) cells. These germline mutated models do not accurately model the sequential somatic Trp53 mutations that arise in Jak2V617F MPN, or track their clonal expansion in vivo. Here, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice to generate a novel mouse model of high molecular risk MPN that transforms to AML. We isolated lineage -/low Sca1 +c-Kit + (LSK) cells from BM of donor mice expressing a conditional allele of mutant Jak2 from its endogenous locus ( Jak2fl-V617F) in combination with an inducible Cas9-IRES-GFP allele and CreER-fusion allele, both from the Rosa26 locus ( Rosa26CreER/lsl-Cas9-IRES-GFP). LSKs were transduced with lentivirus expressing a sgRNA targeting exon 4 of Trp53 (sgTrp53), located in the DNA-binding domain of Trp53. These transfected sgTrp53 LSKs, along with control groups transduced with either empty vector (EV) or a sgRNA targeting Catsper1 (sgCatsper1, off target cutting control), were transplanted into lethally irradiated recipients. Successful engraftment and CreER activity, induced by a 2-week period of Tamoxifen chow, were confirmed by flow cytometry. Validation of Trp53-editing was performed using amplicon sequencing, revealing the most common mutations were frameshift mutations. A subset of these frameshift mutations exhibited an extremely high frequency, conferring a selective advantage within the cell populations in sgTrp53 mice. We further examined the levels of p53 induced by nutlin-3a (an MDM2 inhibitor) in edited BM cells from three groups of mice and found that Trp53 editing leads to a reduction in p53 levels and prevents the loss of viability in response to MDM2 inhibition. This indicates that CRISPR-Cas9 mediated Trp53 mutations result in a loss of function. SgTrp53 mice recapitulated the evolution of leukemic transformation of MPN, showing a PV phenotype for 8 weeks post CreER activation, characterized by elevated hematocrit but normal leukocytes and platelet counts, consistent with the phenotype exhibited by the EV and sgCatsper1 recipients. Subsequently, after 12 weeks, this MPN phenotype transformed to acute leukemia. This disease progression was associated with significantly shortened survival of sgTrp53 recipients, compared to the sgCatsper1 or EV control groups. Histopathologic analysis further supported these findings, showing leukocytosis and the presence of immature blast cells in the PB, spleen and BM of sgTrp53 mice, whereas such cells were absent in the control groups. Notably, the frequency of blast cells was correlated with the frequency of Trp53 frameshift mutations, consistent with what has been reported in MPN patients. Additionally, CRISPR-Cas9 mediated Trp53-loss primarily affected the erythroid lineage, driving expansion of erythroblasts, megakaryocyte-erythroid progenitors (MEP), and Lineage -/low Sca1 -c-Kit + in sgTrp53 mice. The erythroblasts showed aberrant co-expression of CD41 and CD71. Through secondary transplants with unfractionated spleen cells, whole BM cells, or sorted MEP cell populations, we confirmed that the leukemia driven by CRISPR-cas9 mediated Trp53-loss in the Jak2V617F-driven PV is transplantable, and that the MEP compartment in leukemic sgTrp53 mice contains the leukemia initiation population. In total, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice, faithfully replicating the process of clonal expansion and evolution observed in the leukemic transformation of MPN. These mouse models of secondary AML are valuable for testing new therapies targeting disease initiating stem cells. Moreover, our models represent the distinct stages of MPN progression, offering a platform to evaluate preventive and therapeutic interventions at various stages of this disease.

Ppm1d truncating mutations promote the development of genotoxic stress-induced AML.

Hematopoietic stem cells (HSCs) ensure blood cell production during the life-time of an organism, and to do so they need to balance self-renewal, proliferation, differentiation, and migration in a steady state as well as in response to stress or injury. Importantly, aberrant proliferation of HSCs leads to hematological malignancies, and thus, tight regulation by various tumor suppressor pathways, including p53, is essential. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and promotes cell survival upon induction of genotoxic stress. Truncating mutations in the last exon of PPM1D lead to the production of a stable, enzymatically active protein and are commonly associated with clonal hematopoiesis. Using a transgenic mouse model, we demonstrate that truncated PPM1D reduces self-renewal of HSCs in basal conditions but promotes the development of aggressive AML after exposure to ionizing radiation. Inhibition of PPM1D suppressed the colony growth of leukemic stem and progenitor cells carrying the truncated PPM1D, and remarkably, it provided protection against irradiation-induced cell growth. Altogether, we demonstrate that truncated PPM1D affects HSC maintenance, disrupts normal hematopoiesis, and that its inhibition could be beneficial in the context of therapy-induced AML.

Cell fate determinant Llgl1 is required for propagation of acute myeloid leukemia

Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members are conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Inactivation of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in the leukemia stem cell compartment. Re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions.

LMO2 promotes the development of AML through interaction with transcription co-regulator LDB1

One of the characteristics of leukemia is that it contains multiple rearrangements of signal transduction genes and overexpression of non-mutant genes, such as transcription factors. As an important regulator of hematopoietic stem cell development and erythropoiesis, LMO2 is considered an effective carcinogenic driver in T cell lines and a marker of poor prognosis in patients with AML with normal karyotype. LDB1 is a key factor in the transformation of thymocytes into T-ALL induced by LMO2, and enhances the stability of carcinogenic related proteins in leukemia. However, the function and mechanism of LMO2 and LDB1 in AML remains unclear. Herein, the LMO2 gene was knocked down to observe its effects on proliferation, survival, and colony formation of NB4, Kasumi-1 and K562 cell lines. Using mass spectrometry and IP experiments, our results showed the presence of LMO2/LDB1 protein complex in AML cell lines, which is consistent with previous studies. Furthermore, in vitro and in vivo experiments revealed that LDB1 is essential for the proliferation and survival of AML cell lines. Analysis of RNA-seq and ChIP-Seq results showed that LDB1 could regulate apoptosis-related genes, including LMO2. In LDB1-deficient AML cell lines, the overexpression of LMO2 partially compensates for the proliferation inhibition. In summary, our findings revealed that LDB1 played an important role in AML as an oncogene, and emphasize the potential importance of the LMO2/LDB1 complex in clinical treatment of patients with AML.

Spi1 R235C point mutation confers hypersensitivity to radiation-induced acute myeloid leukemia in mice

Ionizing radiation (IR) is a risk factor for acute myeloid leukemia (rAML). Murine rAMLs feature both hemizygous chromosome 2 deletions (Del2) and point mutations (R235) within the hematopoietic regulatory gene Spi1. We generated a heterozygous CBA Spi1 R235 mouse (CBASpm/+) which develops de novo AML with 100% incidence by ∼12months old and shows a dose-dependent reduction in latencyfollowing X-irradiation. These effects are reduced on an AML-resistant C57Bl6 genetic background. CBASpm/Gfp reporter mice show increased Gfp expression, indicating compensation for Spm-induced Spi1 haploinsufficiency. Del2 is always detected in both de novo and rAMLs, indicating that biallelic Spi1 mutation is required for AML. CBASpm/+ mice show that a single Spm modification is sufficient for initiating AML development with complete penetrance, via the "two-hit" mechanism and this is accelerated by IR exposure. Similar SPI1/PU.1 polymorphisms in humans could potentially lead to enhanced susceptibility to IR following medical or environmental exposure.

SETBP1 is dispensable for normal and malignant hematopoiesis.

SETBP1 is a potential epigenetic regulator whose hotspot mutations preventing proteasomal degradation are recurrently detected in myeloid malignancies with poor prognosis. It is believed that the mutant SETBP1 exerts amplified effects of wild-type SETBP1 rather than neomorphic functions. This indicates that dysregulated quantitative control of SETBP1 would result in the transformation of hematopoietic cells. However, little is known about the roles of endogenous SETBP1 in malignant and normal hematopoiesis. Thus, we integrated the analyses of primary AML and healthy samples, cancer cell lines, and a newly generated murine model, Vav1-iCre;Setbp1fl/fl. Despite the expression in long-term hematopoietic stem cells, SETBP1 depletion in normal hematopoiesis minimally alters self-renewal, differentiation, or reconstitution in vivo. Indeed, its loss does not profoundly alter transcription or chromatin accessibilities. Furthermore, although AML with high SETBP1 mRNA is associated with genetic and clinical characteristics for dismal outcomes, SETBP1 is dispensable for the development or maintenance of AML. Contrary to the evidence that SETBP1 mutations are restricted to myeloid malignancies, dependency on SETBP1 mRNA expression is not observed in AML. These unexpected results shed light on the unrecognized idea that a physiologically nonessential gene can act as an oncogene when the machinery of protein degradation is damaged.

MiR-409–3p regulates the proliferation and apoptosis of THP-1 through targeting Rab10

Acute myeloid leukemia cytogenetics and molecular subtypes are connected with microRNAs, although it is unclear how miRNAs affect AML pathogenesis. miR-409–3p expression is downregulated in bone marrows, as we have previously demonstrated in our team. Nevertheless, the tumor-suppressing activities and molecular mechanisms of miR-409–3p remain unknown. Hence, in this study, we investigate at the functional significance of miR-409–3p in the development of AML. We found that a significant decrease in miR-409–3p expression was observed in THP-1 cell. The expression of miR-409–3p was altered in THP-1 by transfecting with agomiR-409–3p and agomiR-409–3p NC. A series of experiments showed that overexpression of miR-409–3p expression significantly suppressed proliferation and increased the apoptosis of THP-1. Moreover, Rab10 was confirmed as a direct target gene of miR-409–3p and was negatively modulated by miR-409–3p. Rab10 downregulation imitated the suppressed proliferation and increased the apoptosis of THP-1. Furthermore, miR-409–3p overexpression or Rab10 knockdown obviously down-regulated the expression levels of Bcl-2, but up-regulated Bax expression. In a xenograft mouse model, miR-409–3p-overexpressed THP-1 cells resulted in much less tumor weight and size in the mice bearing the cells as compared to the mock-transfected mice. Collectively, our findings demonstrated that miR-409–3p exerted tumor suppressor gene effects in AML by directly targeting Rab10, which might provide a promising therapeutic target for AML.

The impact of the ERCC2 Lys751Gln polymorphism on the risk of acute myeloid leukemia in an Iraqi patients

Objectives: AML is the only type of acute leukemia diagnosed in adults and is less common in children. It has the lowest survival rate. This study aimed to investigate the epidemiological risk factors for AML expansion comprise environmental factors, for instance, smoking , and therapy-related factors. Methods :The study was conducted on 70 acute myeloid leukemia patients—37 females and 33 males and on 30 healthy people—12 females and 18 males—as a control group. DNA was extracted from the study groups' whole blood samples using the gSYNCTM DNA Extraction Kit. The T751G polymorphism of the ERCC2 gene was determined by the PCR-RFLP technique. Results: In genetic analysis, it was shown that the carriers of allele Lys and genotype Lys/Lys have a lower risk of developing AML, while allele carriers Gln have an increased risk. The results showed the ERCC2 gene, Lys 751 Gln (T/G) heterozygous TG genotypes, and the G allele were significantly higher (P<0.05) in AML patients compared to the control group. In the sequencing of the region we studied, it was found that there is a site of diversity that is located between the CTTCAG and CTGCAG, where a change in nucleotides (T to G) represents the restriction site of the restriction enzyme. Conclusion : The polymorphic marker 751 Gln> Lys of the ERCC2 gene was associated with the development of AML in Iraqi patients. It was discovered that allele Lys genotype Lys/Lys carriers have a lower risk of developing AML, whereas allele Gln carriers have an increased risk.

A transient receptor potential channel-related model based on machine learning for evaluating tumor microenvironment and immunotherapeutic strategies in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy. Transient receptor potential (TRP) channels in AML still need to be further explored. A TRP channel-related model based on machine learning was established in this study. The data were downloaded from TCGA-LAML and Genome-Tissue Expression (GTEx). TRP-related genes (TRGs) were extracted from previous literature. With the use of Single-Sample Gene Set Enrichment Analysis (ssGSEA), TRP enrichment scores (TESs) were calculated. The limma package was used to identify differentially expressed genes (DEGs), and univariate Cox regression analysis was performed to identify prognostic DEGs. The above prognostic DEGs were analyzed by Random Survival Forest and least absolute shrinkage and selection operator (Lasso) analysis to create the TRP signature. The Kaplan-Meier and receiver operating characteristic (ROC) curves were plotted to investigate the efficiency and accuracy of prognostic prediction. Moreover, genomic mutation analysis was based on GISTIC analysis. Based on ESTIMATE, TIMER, MCPcounter, and ssGSEA, the tumor microenvironment and immunological characteristics were expressly evaluated to explore immunotherapeutic strategies. Enrichment analysis for TRP signature was based on the Kyoto Encyclopedia of Genes Genomes (KEGG), Gene Ontology (GO), over-representation analysis (ORA), and Gene Set Enrichment Analysis (GSEA). Genomics of Drug Sensitivity in Cancer (GDSC) and pRRophetic were used to carry out drug sensitivity analysis. Conclusively, SCHIP1 was randomly selected to perform in vitro cyto-functional experiments. The worse clinical outcomes of patients with higher TESs were observed. There were 107 differentially expressed TRGs identified. Our data revealed 57 prognostic TRGs. Eight TRGs were obtained to establish the prognostic TRP signature, and the worse clinical outcomes of patients with higher TRP scores were found. The efficiency and accuracy of TRP signature in predicting prognosis were confirmed by ROC curves and five external validation datasets. Our data revealed that the mutation rates of DNMT3A, IDH2, MUC16, and TTN were relatively high. The level of infiltrating immune cell populations, stromal, immune, and ESTIMATE scores increased as the TRP scores increased. Nevertheless, AML patients with lower TRP scores exhibited more tumor purity. The TRP scores were found to be correlated with immunomodulators and immune checkpoints, thus revealing immune characteristics and immunotherapeutic strategies. The IC50 values of six chemotherapeutics were lower in the high TRP score (HTS) group. Finally, it was found that SCHIP1 may be the oncogenic gene. The results of this study will help in understanding the role of TRP and SCHIP1 in the prognosis and development of AML.

Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a fatal clonal hematopoietic malignancy, which results from the accumulation of several genetic aberrations in myeloid progenitor cells, with a worldwide 5-year survival prognosis of about 30%. Therefore, the development of more effective therapeutics with novel mode of action is urgently demanded. One common mutated gene in the AML is the DNA-methyltransferase DNMT3A whose function in the development and maintenance of AML is still unclear. To specifically target “undruggable” oncogenes, we initially invented an RNAi-based targeted therapy option that uses the internalization capacity of a colorectal cancer specific anti-EGFR-antibody bound to cationic protamine and the anionic siRNA. Here, we present a new experimental platform technology of molecular oncogene targeting in AML.MethodsOur AML-targeting system consists of an internalizing anti-CD33-antibody–protamine conjugate, which together with anionic molecules such as siRNA or ibrutinib-Cy3.5 and cationic free protamine spontaneously assembles into vesicular nanocarriers in aqueous solution. These nanocarriers were analyzed concerning their physical properties and relevant characteristics in vitro in cell lines and in vivo in xenograft tumor models and patient-derived xenograft leukemia models with the aim to prepare them for translation into clinical application.ResultsThe nanocarriers formed depend on a balanced electrostatic combination of the positively charged cationic protamine-conjugated anti-CD33 antibody, unbound cationic protamine and the anionic cargo. This nanocarrier transports its cargo safely into the AML target cells and has therapeutic activity against AML in vitro and in vivo. siRNAs directed specifically against two common mutated genes in the AML, the DNA-methyltransferase DNMT3A and FLT3-ITD lead to a reduction of clonal growth in vitro in AML cell lines and inhibit tumor growth in vivo in xenotransplanted cell lines. Moreover, oncogene knockdown of DNMT3A leads to increased survival of mice carrying leukemia patient-derived xenografts. Furthermore, an anionic derivative of the approved Bruton’s kinase (BTK) inhibitor ibrutinib, ibrutinib-Cy3.5, is also transported by this nanocarrier into AML cells and decreases colony formation.ConclusionsWe report important results toward innovative personalized, targeted treatment options via electrostatic nanocarrier therapy in AML.

Prospective Evaluation of Sorafenib Combined with Chemotherapy in Newly Diagnosed Adult Core-Binding Factor Acute Myeloid Leukemia: An Open-Label, Randomized Controlled, Multicenter Phase II Trial

Objective Core-binding factor acute myeloid leukemia (CBF-AML) includes AML with t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22) chromosomal rearrangements, leading to the RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes, respectively. Although CBF-AML is considered to have a favorable prognosis relative to other AML subtypes, up to 40% of these patients experience relapse. Recent studies demonstrate achievement of complete molecular remission (CMR) at the end of treatment significantly associated with a reduced risk of relapse. A high frequency of mutations and/or high expression of KIT gene is observed in CBF-AML. Sorafenib is a first-generation type-II multitargeted tyrosine kinase receptor inhibitor (TKI) that suppresses various signaling pathways associated with the development of AML, such as RTK (FLT3, c-KIT). Thus the purpose of this study is to evaluate the safety and efficacy of sorafenib combined with chemotherapy in adult newly diagnosed CBF-AML. Methods Patients were randomly assigned (1:1) to control group (chemotherapy alone) or sorafenib group (sorafenib combined with chemotherapy). For control group, patients received one cycle of induction therapy with idarubicin (12 mg/m² on days 1-3) plus cytarabine (100 mg/m² on days 1-7), followed by one cycle of idarubicin (8 mg/m² on days 1-3) plus cytarabine (2 g/m² q12h on days 1-3) and two cycles of high-dose cytarabine consolidation therapy (2 g/m² q12h on days 1-3). For sorafenib group, patients were treated with chemotherapy plus sorafenib at 400 mg twice daily on days 8-21 for induction cycle, and on days 1-21 for each consolidation, and as maintenance for 12 months. Allogeneic or autologous HSCT were implemented based on the measurable residual disease (MRD) level and donor availability after 4 cycles of therapy. MRD levels were serially monitored for RUNX1-RUNX1T1 or CBFB-MYH11 transcripts by real-time quantitative polymerase chain reaction. Results were expressed as a [fusion gene/ABL1] ×100 transcript ratio. Major molecular response (MMR) and complete molecular remission (CMR) are defined as transcript ratio < 0.1% and 0.001%, respectively. The primary endpoint was CMR after 4 cycles of therapy in bone marrow (BM). Results Between January 2020 and March 2022, 64 patients were enrolled and randomly assigned to sorafenib group (n=32) or control group (n=32). There is no significant difference between the two groups about age, gender, CBF subtype, BM blast percentage, additional chromosome abnormalities and gene mutations at diagnosis. The hematological complete remission in the sorafenib and control group were 96.9% and 94.1%, respectively (p=.592). Following three cycles of therapy, MMR was observed in 79.3% of the sorafenib group and 46.9% of the control group (p =.009). Sorafenib group also had a significantly higher CMR than control group (62.1% vs 28.1%, p =.009). After four cycles of therapy, MMR increased to 100% in the sorafenib group and 70.8% in the control group (p =.019), whileCMR was also more frequently achieved in the sorafenib group than that in the control group (90.9% vs 54.2%, p =.006). There was no significant difference of grades 3-4 adverse events between the two groups. Grade 4 neutropenia (neutrophil count less than 0.5×10⁹ cells per L) and thrombocytopenia (platelet count less than 20×10⁹ per L) were observed in all cases. The median duration of neutropenia was 12d (range, 6-25d) in the sorafenib group and 10d (range, 5-22d) in the control group (p =.063) , while the median duration of thrombocytopenia was 12d (range, 5-33d) and 9d (range, 6-19d) (p =.052), respectively.The most common grade 3-4 non-hematological adverse events in sorafenib and control group were pneumonia (31.3% vs 57.4%, p=.019), mucositis (21.9% vs 24.1%, p =.816), blood stream infection (37.5% vs 31.5%, p =.568). Conclusion Sorafenib combined with chemotherapy significantly increased the CMR after 4 cycles of therapy, however, the incidence of haematological and non-haematological adverse events did not apparently increased. The impact of sorafenib combined with chemotherapy on relapse and survival of CBF-AML need further following-up. Keywords: Sorafenib, CBF-AML, CMR, adverse events

Identification of a Novel FAM135B Cancer Susceptibility Mutation in a Family with Multiple Cases of MLL-Rearranged AML

Background: Familial aggregation of malignancies, including leukemia, is rarely reported due to the scattered cases, weakened bonding among generations, and low birth rates in modern society. The emerging demands for predicting and preventing cancer development are outgrowing the progress in the studies of the mechanism underlying the propensity of malignancies. Results: Here we reported a family with 4 cases of acute leukemia (AL) and four solid tumors (2 for lung cancer, 1 for renal clear cell carcinoma, and one for rectal adenocarcinoma). Besides, one member in the family was diagnosed with intraspinal cavernous hemangioma, and another one was diagnosed with congenital heart disease (transposition of the great arteries). 2 out of 4 AL patients were cousins and diagnosed with acute monocytic leukemia (AML-M5 with MLL-rearrangement) in our center. Whole genome sequencing was performed on two patients with AML, two patients with solid tumors, one obligated carrier, and three healthy members. Using an in-house bioinformatic data analysis pipeline, we identified 18 single nucleotide variants (SNVs) and four insertions/deletions (Indel) in all the affected members and one obligated carrier while absent in healthy members. Genotyping of these variants in 57 family members confirmed that 18/22 variants co-segregated with the malignant phenotype of this family. By screening for these 18 variants in 13 AL pedigrees, we narrow our cancer-predisposing candidate list to one variant, hitting FAM135B (family with sequence similarity 135 member B; NM_015912: exon13:c.A2621G:p.E874G), which is recurrent in another AML pedigree with two cases in two consecutive generations. This mutation resides in a highly conserved locus among species and is predicted to be deleterious. FAM135B is predicted to encode serine esterase, and the role of FAM135B in AML was not documented. Intriguingly, all the affected familial AML cases from two pedigrees were diagnosed with AML-M5 with MLL-rearrangement. Moreover, this mutation is recurrent in sporadic adult AML cohort (6/467), among which half (3/6) were AML-M5, two M3, and one M2. Functionally, we found that overexpression of FAM135B promotes the proliferation and colony formation of AML cells. Consistently, downregulation of FAM135B nearly abolished the colony-forming capacity of AML cells and impaired cell proliferation. Further studies on the role of FAM135B in AML development are under investigation. Conclusion: We identify a novel cancer-predisposing gene, FAM135B, and its mutation E874G occurred in both familial and sporadic AML cases, especially AML-M5 with MLL-rearrangement.

A Phase II Study of Mini-Hyper-CVD Plus Inotuzumab Ozogamicin, with or without Blinatumomab, in Older Adults with Newly Diagnosed Philadelphia Chromosome-Negative B-Cell Acute Lymphoblastic Leukemia: Updated Results and Predictors for Outcomes

Background: Inotuzumab ozogamicin (INO) and blinatumomab are superior to chemotherapy for relapsed/refractory B-cell acute lymphoblastic leukemia (ALL). Incorporation of these agents in the frontline setting could improve remission durations and OS in older adults with newly diagnosed B-cell ALL. Methods: Patients (pts) ≥60 years of age with newly diagnosed Philadelphia chromosome (Ph)-negative pre-B-cell ALL were eligible. Pts were required to have a performance status of ≤3, total bilirubin ≤1.5 mg/dl, AST/ALT ≤3x ULN and creatinine ≤2 mg/dl. Pts received mini-hyper-CVD (cyclophosphamide and dexamethasone at 50% dose reduction, no anthracycline, methotrexate at 75% dose reduction, cytarabine at 0.5 g/m2 x 4 doses) for up to 8 cycles. INO was given at a dose of 1.3-1.8mg/m2 on day 3 of cycle 1 and 0.8-1.3mg/m2 on day 3 of cycles 2-4. Rituximab (if CD20+) and prophylactic IT chemotherapy were given for the first 4 cycles. Responding pts received POMP maintenance for up to 3 years. To decrease the risk of veno-occlusive disease (VOD), the protocol was amended in 3/2017 (pts 50+) to give INO in fractionated doses each cycle (0.6 mg/m2 on day 2 and 0.3 mg/m2 on day 8 of cycle 1; 0.3 mg/m2 on day 2 and 8 of cycles 2-4) and to administer 4 cycles of blinatumomab following 4 cycles of hyper-CVD plus INO, followed by maintenance with 12 cycles of POMP and 4 cycles of blinatumomab (1 cycle of blinatumomab after every 3 cycles of POMP). The cumulative dose of INO given before and after this most recent amendment was 4.3 mg/m2 and 2.7 mg/m2, respectively. Results: To date, 80 pts have been treated. Six pts were in complete remission (CR) at the time of enrollment. Baseline characteristics are shown in Table 1. The median age was 68 years (range, 60-87 years); 30 pts (39%) were ≥70 years. From all pts, 39% were positive for TP53 mutation and 24% had an adverse-risk karyotype. Among 74 pts evaluable for morphologic response, 73 (99%) responded (CR, n=66; CRp, n=6; CRi, n=1). MRD negativity by flow cytometry was achieved in 61/76 pts (80%) after 1 cycle and 74/79 pts (94%) overall. The 30-day and 60-day mortality rates were 0% and 3%, respectively. Among 79 pts who achieved remission, 11 (14%) relapsed, 4 (5%) underwent allogeneic SCT in first remission (1 of whom subsequently relapsed), 32 (40%) are alive in remission without SCT, and 32 (40%) died in remission. Among the 32 deaths in remission, 9 were from sepsis, 3 from VOD, 9 from development of MDS or AML; the rest from miscellaneous causes. Overall, 9 pts (11%) developed MDS/AML after a median of 33 months (range 3-66); 7 of these pts had a detectable TP53 mutation in the myeloid malignancy. Notably, 6 pts (8%) developed VOD, 1 after subsequent allogeneic SCT. With a median follow-up of 61 months (range, 7-123 months), the 5-year CR and OS rates were 76% and 46%, respectively. Outcomes were superior in pts who were 60-69 years old versus those who were ≥70 years (5-year OS rates: 57% and 28%, respectively; P=0.05) and for those without poor-risk cytogenetics (e.g. KMT2A-rearranged, low hypodiploidy/near triploidy, complex cytogenetics) versus poor-risk cytogenetics (5-year OS rates: 55% and 21%, respectively; P=0.005). The 5-year OS for pts with and without TP53 mutation were 35% and 47%, respectively (P=0.34). The 5-year OS for pts age 60-64 was 65%, for those age 65-69 was 50%, and for those age ≥70 was 28%. The 3-year OS for patients with and without CRLF2+ ALL were 25% and 60%, respectively (P=0.12). Pts ≥70 years of age were more likely to die in remission (19/30 [63%]) and those who were 60-69 years of age (15/50 [30%]) (P=0.004), which was a major driver of the poor survival in this age group. Outcomes were favorable for pts age 60-69 years with poor risk cytogenetics (5-year OS 70%), intermediate for pts age 60-69 with poor risk cytogenetics (5-year OS 31%) or those age ≥70 with low risk cytogenetics (5-year OS 36%), and were very poor for pts age ≥70 with poor risk cytogenetics (5-year OS 0%) (Figure 1). Conclusion: Low-intensity chemotherapy with hyper-CVD plus INO, with or without blinatumomab, in older adults with newly diagnosed Ph-negative ALL resulted in very high rates of response (99%), including an MRD-negative response rate of 94%. This led to a 5-year OS rate of 46%. Despite the reduced intensity of this regimen, a substantial proportion of pts >70 years of age died in remission. Ongoing efforts are evaluated for INO and blinatumomab without chemotherapy for these pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Oncogene EVI1 Drives Acute Myeloid Leukemia Via a Targetable EVI1-CtBP1/2 Interaction and By Repressing Myeloid Regulator CEBPA

Overexpression of EVI1 (located at 3q26), as a result of 3q26 rearrangements, causes the development of one of the most aggressive, chemotherapy-resistant forms of AML. We hypothesize that insight into the function of EVI1 in AML will lead to the identification of targetable vulnerabilities for those patients. EVI1 encodes a DNA-binding zinc-finger protein that has a critical role in transcriptional regulation and maintenance of quiescent hematopoietic stem cells. Here, we aim to understand the mechanism by which EVI1 overexpression in bone marrow progenitors drives AML development. To identify its target genes, we made EVI1 rapidly degradable by endogenous fusion with an auxin-inducible degron (AID) tag in inv(3q21;3q26) rearranged MUTZ3 leukemia cells. Degradation of EVI1 strongly increased myeloid differentiation of MUTZ3 cells. Using metabolic labeling of newly produced RNA (SLAM-seq), we found 6 genes to be upregulated after 1.5 hrs of degradation. Out of those genes, CEBPA was also upregulated upon shRNA knockdown of EVI1 or after introducing inactivating mutations in the hijacked GATA2 enhancer that drives EVI1 expression. Those findings suggest persistent upregulation of CEBPA when EVI1 levels are reduced. In line with this, CEBPA expression is 1.83 fold (p=0.00024) lower in AML patients with 3q26 rearrangements (n=18) compared to non-3q26 rearranged cases (n=97). Interestingly, overexpression of CEBPA in MUTZ3 cells bypassed EVI1 driven repression and caused myeloid differentiation as well. We next addressed the question how EVI1 represses CEBPA transcription. The CEBPA locus contains 14 enhancers in a 100kb region around the gene body, among which the myeloid specific +42kb enhancer, essential for neutrophil development. EVI1 binds this +42kb enhancer of CEBPA as determined by chromatin immunoprecipitation sequencing (ChIP-seq) in 3q26-rearranged AML and MUTZ3 cells. After degradation of EVI1, H3K27Ac levels increased at this enhancer, indicating that EVI1 reduces activity of the +42kb CEBPA enhancer. Since EVI1 lacks any enzymatic domains that could modulate chromatin, we sought to further understand how EVI1 represses activity of the +42kb CEBPA enhancer. EVI1 protein immunoprecipitation followed by mass spectrometry revealed interaction with CtBP1/2, factors known to interact with repressors such as Polycomb proteins or HDACs. ChIP-seq showed a strong overlap between CtBP2 and EVI1 binding sites in MUTZ3 cells. Moreover, EVI1 elimination caused loss of CtBP2 binding to the chromatin including to the +42kb CEBPA enhancer. EVI1 harbors a PLDLS domain essential for binding to CtBP1/2. Mutation of this domain abolished CtBP2 binding and reduced transformation of mouse bone marrow progenitors by EVI1, as determined by in vitro replating assays. We reasoned we could exploit the high affinity of EVI1 for CtBP through this single domain by overexpressing a repeat of a small section of EVI1 containing only the CtBP-binding PLDLS-site. Overexpression of "4X-PLDLS competitor peptide" was able to sequester CtBP and thereby prevent it's binding to EVI1. The competitor peptide was able to abolish in vitro replating capacity and re-enabled differentiation of mouse bone marrow progenitors transformed with EVI1. In line with this, transduction of the competitor peptide into MUTZ3 cells led to upregulation of CEBPA as well as growth inhibition. Furthermore, in vivo leukemia outgrowth in a xenotransplant model of 3q26 rearranged AML cell lines MUTZ3 and SB1690CB was fully abolished by the competitor peptide, but not by a mutant control peptide. In conclusion, EVI1 is a repressor of active chromatin regions, including CEBPA via the +42kb myeloid specific enhancer. Repression by EVI1 is dependent on its interaction with CtBP1/2, proteins that recruit repressors of transcription via an essential . A PLDLS domain in EVI1 is essential for this interaction. The competitor peptide experiments point to exploiting this interaction with repressive molecules as a potential therapeutic target in AML with overexpression of EVI1.