PI3Kδ Inhibition Suppresses Central Nervous System Involvement of Acute Lymphoblastic Leukemia

Abstract The majority of children and adults who experience relapse of acute lymphoblastic leukemia (ALL) will die from the disease. The presence of leukemic blasts within the cerebrospinal fluid (CSF) is an important predictor of disease recurrence in both the bone marrow (BM) and central nervous system (CNS), a frequent site of disease involvement. In the absence of intrathecal chemotherapy or craniospinal irradiation, 30%‒50% of patients will in fact develop CNS disease. All patients therefore receive CNS prophylaxis. High-risk patients require intensive CNS-directed treatment that causes toxic side effects and can impact cognitive development in children. Patients who develop recurrence in the CNS have limited treatment options and an extremely poor prognosis. The enzyme PI3K plays an important role in many biological effects including cell proliferation, migration, and apoptosis. The d isoform of PI3K is specifically expressed in immune cells. The PI3Kd inhibitor idelalisib is approved for use in combination with rituximab for the treatment of chronic lymphocytic leukemia (CLL). However, little is known about the efficacy of PI3Kd inhibition in ALL. Here, the PI3Kd inhibitor GS-649443, a potent and specific in vivo tool compound, was evaluated in a xenograft mouse model. The in vivo effects of GS-649443 were investigated in a Nalm-6 pre-B ALL SCID model. At approximately 40 days postengraftment, untreated mice all developed symptoms of CNS involvement and displayed hind limb paralysis. Hind limb paralysis is the clinical endpoint for sacrifice, occurring prior to death from progressive BM disease. Mice were treated with GS-649443 by oral gavage beginning on postengraftment day 1 and continuing until the development of clinical symptoms requiring sacrifice (hind limb paralysis, weight loss >20%, respiratory or other distress). Only 17% of GS-649443‒treated mice developed hind limb paralysis at their clinical endpoint, while 100% of vehicle-treated mice showed hind limb paralysis. In addition, mice treated with GS-649443 or vehicle control were paired. When either mouse in a treatment pair reached a clinical endpoint, both were sacrificed so disease burden in individual organs could be compared at matched time points. There was no difference in tumor burden or leukemic cell apoptotic rate in the BM of treated vs vehicle control groups. In contrast, there was a significant decrease in the number of leukemic blasts harvested from the CSF of treated mice (Fig. 1). Consistent with the inhibition of CNS disease progression, GS-649443 significantly prolonged the survival of treated mice (Fig. 2). While compromised blood-brain barrier in leukemic mice may allow therapeutic targeting of CNS disease, it is unknown whether GS-649443 enters the CNS in healthy rodents. Given the well-described effects of idelalisib on CLL cell migration, we hypothesized that PI3Kd blockade impacts disease in this ALL model by impairing leukemia migration into the CNS. To investigate the in vitro effect of GS-649443 on ALL cell migration, transwell migration assays with SDF-1 as chemoattractant were performed. Both GS-649443 and idelalisib suppressed migration of Nalm-6 and primary human ALL cells toward SDF-1. Lastly, the effects of GS-649443 in combination with conventional chemotherapy were examined. GS-649443 alone did not cause significant cytotoxicity of Nalm-6 in vitro, however, treatment with GS-649443 after cytarabine significantly increased apoptosis. Leukemic mice (20 days post-engraftment) were then treated with cytarabine (days 1‒5) in combination with GS-649443 or cytarabine alone (days 1‒7) for 1 cycle. In contrast to the single-agent study, significant decreases in the number of leukemic blasts harvested from both the BM and CSF of treated mice were observed, suggesting that PI3Kd inhibition sensitized ALL cells to cytarabine chemotherapy (Fig. 3). This decrease in residual disease after combination therapy led to significantly prolonged survival in GS-649443‒treated mice (Fig. 4). Taken together, this study provides evidence that PI3Kd inhibition prevents ALL progression in the CNS. In addition, GS-649443 and conventional chemotherapy appear to synergize to decrease residual BM disease. The potential for PI3Kd inhibition to improve chemotherapy response and impede development of CNS disease in ALL warrants further investigation in a translational clinical study. Disclosures Yao: Gilead Sciences, Inc.: Research Funding. Price:Gilead Sciences, Inc.: Research Funding. Olivere:Gilead Sciences, Inc.: Research Funding. Warner:Gilead Sciences, Inc.: Research Funding. Tannheimer:Gilead Sciences: Employment. Sipkins:Gilead Sciences, Inc.: Research Funding.

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The HB22.7 Anti-CD22 Monoclonal Antibody Is an Effective Platform for CD22-Targeted Antibody Drug Conjugates for Treatment of Lymphoma and Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1653.1653 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1653-1653

Author(s):

Soames F. Boyle ◽

Jason Kato ◽

Robert O'Donnell ◽

Joseph Tuscano

Keyword(s):

Monoclonal Antibody ◽

Acute Lymphoblastic Leukemia ◽

Hind Limb ◽

Lymphoblastic Leukemia ◽

Raji Cells ◽

Drug Conjugates ◽

Hind Limb Paralysis ◽

Antibody Drug

Abstract Abstract 1653 Many non-Hodgkin lymphomas (NHL) and acute lymphoblastic leukemia (ALL) initially respond to therapy, but relapse; at that point further treatment is often limited by chemotherapy-related toxicity. CD22 is a valid target for immunotherapy of NHL and ALL. We have shown that the anti-CD22 ligand blocking monoclonal antibody (mAb), HB22.7, has significant activity. Antibody drug conjugates (ADC) are emerging as the next generation of targeted therapies for lymphoid neoplasms as they may be designed to be effective but with manageable toxicity. We sought to determine if HB22.7 could be used as a platform for CD22-targeted ADCs for the treatment of lymphoid neoplasms. We describe here the effect H22.7-saporin (SAP), a novel ADC composed of HB22.7 and a ribosome inactivating protein on ALL and NHL models. The in vitro cytotoxicity of HB22.7-SAP was assessed using two pre-B cell ALL lines REH and JM1, and NHL cell lines Ramos, Raji, Granta 519, SU-DHL-4, and DOHH-2. HB22.7-SAP had IC50 values of 0.7 and 1.2 ng/mL in ALL and 1.0–8.4 ng/mL in the NHL cell lines. Free HB22.7 plus SAP did not have any cytotoxic effect using the same doses. HB22.7-SAP was not cytotoxic to CD22 negative Jurkat cells. To evaluate HB22.7-SAP's in vivo efficacy against a model of human ALL, NOD/SCID mice were injected with REH leukemia cells. Twenty-four hours later the mice were treated with PBS, free HB22.7 plus SAP, or HB22.7-SAP. Treatment was continued twice weekly for 4 weeks. Mice were euthanized at the onset of hind limb paralysis. The median survival was 52 days as compared to 20 days for the untreated mice (p < 0.0005). Mice treated with HB22.7-SAP had normal blood counts over the study period. In contrast, mice from the PBS or free HB22.7 plus free SAP groups developed leukopenia and thrombocytopenia by day 20, coincident with development of ALL; bone marrow from the two mice that developed hind limb paralysis confirmed ALL. To assess efficacy in a human model of NHL, Raji cells were injected into the flank of previously radiated, female, athymic nude mice. Tumors grew until they reached 100–200 mm3 (day 0). Mice were then exposed to HB22.7-SAP, PBS, or free HB22.7 plus free SAP. Each treatment was administered intraperitoneally, weekly for 3 weeks. To assess significance of starting tumor volume on HB22.7-SAP-mediated tumor growth inhibition, a second model initiated therapy 24 hours after tumor implantation and used the same dose and drug schedule. During the first 14 days, HB22.7-SAP (1 mg/kg) significantly inhibited tumor growth (p < 0.01) on days 5 and 8 as compared to the PBS and free HB22.7 plus free SAP control groups, however, by day 19, the average tumors volume of mice treated with HB22.7-SAP were no different than the controls. In contrast, when HB22.7-SAP was administered 24 hours after subcutaneous injection of Raji cells no mice in the treatment group developed measureable tumors in the 50-day study period. There was no difference in hematologic toxicity between the groups however mice treated with HB22.7-SAP had more therapy-related weight loss. This study demonstrates the promising in vivo and in vitro activity of HB22.7 when used as a platform for CD22-targeted ADCs. The HB22.7-SAP ADC is both specific and cytotoxic to CD22 expressing malignant lymphocytes and that translates into significant efficacy against NHL and ALL. When compared to other anti-CD22 mAb we hypothesize that HB22.7-based ADCs will prove to be potent cytotoxic drugs in-part due to their unique ability to block ligand binding thus conferring an independent lymphocidal property. Disclosures: No relevant conflicts of interest to declare.

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AVA4746, an Orally Available, Clinical Grade Antagonist of Integrin Alpha 4, Sensitizes Pre-B Cell Acute Lymphoblastic Leukemia to Chemotherapy

Blood ◽

10.1182/blood.v128.22.2765.2765 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2765-2765 ◽

Cited By ~ 2

Author(s):

Yongsheng Ruan ◽

Eun Ji Gang ◽

Hye-Na Kim ◽

Chintan Parekh ◽

Hisham Abdel-Azim ◽

...

Keyword(s):

Overall Survival ◽

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Metabolic Activity ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Drug Resistant ◽

Support Research

Abstract Background. Even though remarkable progress has been made in the treatment of childhood acute lymphoblastic leukemia (ALL), salvage of relapse patients remains a challenge. The role of the bone marrow (BM) microenvironment is critical to protect leukemia cells from chemotherapy. The BM microenvironment promotes cell adhesion-mediated drug resistance (CAM-DR) in ALL.We and others have shown that the adhesion molecule integrin α4, referred to hereafter as α4, mediates drug resistance of B-ALL. In our previous studies, we showed that both α4 blockade by natalizumab and inhibition by the small molecule α4 antagonist TBC3486 can sensitize relapsed ALL cells to chemotherapy. However, no α4 targeting therapy is currently clinically available to treat leukemia. Here, we preclinically evaluate a novel non-peptidic small molecule antagonist, AVA4746, which has been safely used in clinical studies, as a potential new approach to combat drug resistant ALL. Method. Six refractory or relapsed primary pre-B ALL cases were used for in vitro studies. Viability was assessed by trypan blue counts or annexin V/7AAD flow cytometric analysis and metabolic activity was evaluated by Cytoscan WST-1 assay. For in vivo evaluation a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL (LAX7R) was used.AVA4746 (15mg/kg) was administered by oral gavage twice a day continuously for 14 days, and vincristine, dexamethasone, L-asparaginase (VDL) was given intraperitoneally (weekly) for 4 weeks. Overall survival was determined by Kaplan-Meier Survival analysis. Results. AVA4746 caused a significant decrease in mean fluorescence intensity (MFI) of α4 expression in six out of six ALL cases at doses of both 5μM and 25μM after 24 hours and 96 hours compared to DMSO control. Interestingly, decreased protein expression of α4 was also observed by Western Blot analysis all six ALL cases. We tested next in two cases (LAX53, ICN13), if AVA4746 de-adheres ALL cells from its counter receptor VCAM-1. The percentages of adherence after treatment with AVA4746 (25μM) were significantly lower than after DMSO treatment (10.3%±4.9% vs. 99.9%±7.6%, p= 0.00007 for LAX7R; 8.1%±1.0% vs. 100.1%±13.6%, p= 0.0003 for LAX53; 9.0%±1.6% vs. 100.0%±14.0%, p=0.0004 for ICN13). AVA4746 was not associated with apoptosis in vitro alone or in combination with chemotherapy (VDL). Metabolic activity as assessed by WST-1 assay was markedly decreased by AVA4746 in two of two ALL cases. AVA4746 also decreased ALL proliferation in two out of two ALL samples tested. In vivo, AVA4746 in combination with VDL chemotherapy treatment led to significant prolongation of overall survival (n=6) compared with the VDL only treated group (n=6) (MST= 78.5 days vs MST= 68 days; P<0.05). There was no significant difference in survival between the PBS control group (n=5) and the AVA4746 mono-treatment group (n=5) (MST=38days vs MST= 38days). Conclusion. We have identified α4 as a central adhesion molecule in CAM-DR of ALL and have shown that AVA-4746, an orally available and specific α4 antagonist, which has been safely used in clinical studies, downregulates α4 in primary ALL and functionally de-adheres them from VCAM-1. Critically, we demonstrated that inhibition of α4 in combination with standard chemotherapy can prolong the survival of NSG mice bearing pre-B ALL. These data support further study of inhibition of α4 using AVA4746 as a novel strategy to treat drug resistant B lineage ALL. Disclosures Bhojwani: Amgen: Other: Blinatumumab global pediatric advisory board 2015. Wayne:Spectrum Pharmaceuticals: Honoraria, Other: Travel Support, Research Funding; Kite Pharma: Honoraria, Other: Travel support, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support; Medimmune: Honoraria, Other: Travel Support, Research Funding; NIH: Patents & Royalties. Kim:Antisense Therapeutics Ltd: Patents & Royalties.

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Functional Analysis of the Subclonal Architecture of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) at a Single Cell Level

Blood ◽

10.1182/blood.v124.21.3778.3778 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3778-3778

Author(s):

Bartosch Wojcik ◽

Fabian Lang ◽

Susanne Badura ◽

Anja Vogel ◽

Tamara Tesanovic ◽

...

Keyword(s):

Single Cell ◽

Research Funding ◽

Cell Tracking ◽

Lymphoblastic Leukemia ◽

Leukemic Cell ◽

Cd38 Expression ◽

Cell Fractions

Abstract Introduction: Transforming events in B-lineage (ALL) occur primarily at the level of committed progenitor cells, but the phenotype, frequency and hierarchical organization of leukemia-initiating cells (LICs) are controversial. Pronounced clonal heterogeneity in ALL and the lack of preclinical models impede functional analysis of LICs and dissecting the clonal architecture of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Aims and methods: To identify cellular features associated with cell fate and decisions, engraftment potential, growth kinetics and resistance to therapy of BCP-ALL LICs at the clonal level, we generated multiple single-cell-derived clones from patient derived-long-term cultures (PD-LTCs) of human BCP-ALL. This in vitro ALL-LTC model allows prolonged serum-, cytokine-, and stroma-free culture of patient-derived BCP-ALL cells that are largely stable in terms of cytogenetic and immunophenotypic features for up to 6 months [Nijmeijer B et. al, Exp Hematol. 2009;37:376; Badura S et al., PLoS One. 2013; 8:e80070] Three ALL-LTCs were selected on the basis of different patterns of surface marker expression and cytogenetics, i.e. Ph+ ALL (LTCs PH and BV; common-ALL) and BCR-ABL negative ALL (LTC CR; pre B-ALL), respectively. Results: Clonogenic growth in semisolid medium ranged from 0.25% to 8% for these ALL-LTCs (CR 8%, PH 0.45% and BV 0.25%), consistent with functional diversity within the bulk as well as the immunophenotypically defined subsets. To analyze these subpopulations at the clonal level, we isolated leukemic cell subsets by cell sorting on the basis of CD20, CD34 and CD38 expression, and then generated hundreds of clones by expanding single-cell sorted subpopulations. All sorted cell fractions from all three ALL-LTCs were capable of initiating long-term growth in vitro. In limiting dilution cultures, 1% to 5% of single sorted cells derived from these cell fractions gave rise to prolonged leukemic cell growth. To evaluate the leukemia-initiating capacity of the clonal ALL subpopulations in vivo, we examined engraftment and outgrowth kinetics of different ALL clones in a xenograft model with sublethally irradiated NSG mice. Individual clones derived from different sorted subpopulations displayed pronounced differences in engraftment potential and aggressiveness based on analyses of blood, bone marrow and spleen. This ranged from rapid appearance of ALL and death within 70-80 days (clone PH 20), to complete lack of leukemic outgrowth 155 days after transplantation (clone PH clone 14). The other four clones derived from this ALL (PH) displayed intermediate engraftment and outgrowth kinetics. Notably, leukemogenic properties were not associated with a distinct surface marker profile based in CD20, CD34 and CD38 expression. A similar heterogeneity of leukemogenic potential was determined with clonal subpopulations from the LTC BV. To assess the hierarchical relationship between different clones and the association with LIC capacity, we examined the pattern of immunoglobulin VDJ-rearrangement of different clones. The Ig rearrangement patterns confirm that the clones represent distinct clonal populations derived from the original polyclonal LTC, but we observed no hierarchical relationship with respect to the biological properties of different clones. Further functional evaluation of these subclones was performed by videomicroscopy-based single cell tracking that allows analysis of individual cells and their progeny over many cell divisions. We observed concordance between cell cycle length and cell death events as determined by single cell tracking and the engraftment potential and aggressiveness of LTCs derived single cell clones in vivo. To elucidate the molecular basis for the biologic differences between the clonal ALL populations, we compared highly and poorly leukemogenic clones by SILAC based proteomics. Only 5% of 6500 proteins measured in clones differing in their leukemia-initiating potential were differentially regulated, and these proteins could be assigned to a limited number of pathways. In conclusion, comparative evaluation of clonal lymphoblast populations generated from patient-derived ALL long-term cultures by combined in vitro analysis, single cell videomicroscopy, xenotransplantation and proteomics is a promising approach to identify specific markers and therapeutic strategies that target LIC in ALL. Disclosures Wojcik: Novartis: Research Funding, Travel, Accommodation Other; SAnofi Aventis: Consultancy, Travel, Accommodation, Travel, Accommodation Other. Lang:Novartis: Research Funding, Travel, Accommodation Other. Brüggemann:Amgen Inc.: Consultancy, Research Funding.

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Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Blood ◽

10.1182/blood-2018-99-120120 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1322-1322

Author(s):

Manabu Kusakabe ◽

Ann Chong Sun ◽

Kateryna Tyshchenko ◽

Rachel Wong ◽

Aastha Nanda ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Research Funding ◽

De Novo ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Rna Seq ◽

Cell Acute Lymphoblastic Leukemia

Abstract Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

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Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Blood ◽

10.1182/blood-2020-139625 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-13

Author(s):

Hong Mu-Mosley ◽

Lauren B Ostermann ◽

Ran Zhao ◽

Challice L. Bonifant ◽

Stephen Gottschalk ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Vehicle Control ◽

Advisory Committees ◽

Cellular Therapies

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

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Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-145956 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 615-615

Author(s):

Yuxuan Liu ◽

Lucille Stuani ◽

Dorra Jedoui ◽

Milton Merchant ◽

Astraea Jager ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Bcr Signaling ◽

Proliferation In Vitro

Abstract Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether. To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway. We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism. To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004). Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. Disclosures Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

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Stearoyl-CoA Desaturase (SCD) Enhances Central Nervous System Leukemia

Blood ◽

10.1182/blood-2018-99-114749 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 389-389

Author(s):

Angela Maria Savino ◽

Orianne Olivares ◽

Shani Barel ◽

Lev Yakimov ◽

Ifat Geron ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Hind Limb ◽

Leukemia Cell ◽

Leukemic Cells ◽

Nsg Mice ◽

Cns Relapse ◽

Hind Limb Paralysis

Abstract Background: Central nervous system (CNS) involvement by acute lymphoblastic leukemia (ALL) is a major clinical concern. Leukemic cells can be found in the CNS at diagnosis (1-2%) or, more frequently, at relapse (30%). Very little is known about the pathogenesis and therefore there are no targeted therapies. Prophylactic CNS-directed conventional intrathecal chemotherapy or irradiation are required for relapse-free survival. However, they are associated with substantial rates of short and long term toxicity. Therefore, elucidation of molecular mechanisms and pathways mediating leukemia-cell entry and survival in the CNS is needed to develop alternative CNS-directed treatment strategies. Previous studies showed an increased expression of Stearoyl-CoA desaturase (SCD), a key enzyme of the de novo fatty acid synthesis pathway, in B cell precursor (BCP) ALL cells isolated from cerebrospinal fluid (CSF) of patients at the time of CNS relapse. A small SCD positive population was detected in the bone marrow (BM) at leukemia diagnosis in patients who later developed isolated CNS relapse, defining a potential biomarker for CNS relapse. It is unknown, however, if SCD has a functional role in CNS leukemia. Aim: To examine the hypothesis that increased expression of SCD enhances trafficking and survival of human B-ALL cells in the CNS Methods: We analyzed leukemia-cell entry into the CNS using xenografts of human BCP-ALL cell lines. Microarray profile of cells isolated from CNS and BM of transplanted mice was performed. Cell lines were transduced to overexpress human SCD and evaluated in vitro for proliferation kinetics and metabolic SCD activity. In vivo, SCD overexpressing cells were transplanted in NSG mice,sacrificed upon the first symptoms of CNS involvement, e.g. hind limb paralysis. BM, spleen and meninges were collected and analyzed to check human engraftment by FACS. The tumor load was expressed as total amount of leukemic cells in each organ. Competition assays were performed by transplanting SCD overexpressing and WT cells in the same mouse in a 1:1 ratio. Results: BCP-ALL cells transplanted into NSG mice faithfully recapitulated pathological features of meningeal infiltration seen in patients with ALL. Gene expression analysis of cells collected from BM and meninges of leukemic mice revealed up-regulation of the genes belonging to the signaling pathway of sterol regulatory element binding proteins (SREBPs) in ALL cells isolated from the CNS. SCD, whose transcription is controlled by the SREBP family, was significantly upregulated. SCD overexpression did not alter proliferation in vitro. Since SCD introduces a double bond in Stearoyl-CoA, its activity was measured as the ratio of unsaturated/saturated fatty acids in the cells. That ratio was increased in SCD overexpressing cells in vitro, confirming the functionality of the enzyme. In vivo, mice transplanted with SCD overexpressing cells led to a faster onset of CNS disease manifested by a clinical phenotype of earlier hind limb paralysis compared to control and significant increased number of leukemic cells in the CNS (Figure 1A).SCD overexpression also induced CNS engraftment of another B-ALL cell line, REH, which is not naturally prone to invade the central nervous system. Mice transplanted with SCD overexpressing REH cells showed the same phenotype of earlier hind limb paralysis and accumulation of leukemic cells in the CNS as the CNS-prone 018z cells, while WT REH did not show any CNS engraftment but comparable tumor load in BM and spleen (Figure1B). To reproduce the clonal heterogeneity in SCD expression observed previously in patients' BM, we performed a competition assay transplanting SCD overexpressing cells and control cells, expressing different fluorochromes, in the same mouse in a 1:1 ratio. In the CNS, the ratio between SCD overexpressing and WT cells ranged from 2 to 20 fold. This effect was unique to the CNS and not reproducible in the other hematopoietic organs where the 1:1 ratio was maintained (Figure 1C). Moreover, SCD overexpression sensitized leukemic cells to mTOR inhibitors, suggesting a potential therapeutic option Conclusion: SCD has a role in homing and survival of leukemic cells in the CNS and may be used as early predictor of CNS relapse. This study reveals a role for SCD and fatty acid metabolism in the pathogenesis of CNS leukemia suggesting that this pathway maybe targeted for specific therapy of this devastating disease. Figure 1. Figure 1. Disclosures Halsey: Jazz Pharmaceuticals: Honoraria, Other: Support for conference attendance.

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Alisertib Synergistically Strengthens the Anti-Leukemia Activity of Venetoclax in TCF3-Hlf B-ALL

Blood ◽

10.1182/blood-2021-148671 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 705-705

Author(s):

Asen Bagashev ◽

Joseph Patrick Loftus ◽

Colin Wakefield ◽

Gerald Wertheim ◽

Christian Hurtz ◽

...

Keyword(s):

Research Funding ◽

Clinical Investigation ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Case Series ◽

Multi Drug Resistance ◽

Drug Synergy ◽

Pdx Models

Abstract Background: Despite maximally-intensive chemotherapy and stem cell transplantation, survival of patients with the very rare t(17;19)/TCF3-HLF B-acute lymphoblastic leukemia (B-ALL) subtype remains effectively 0%. Prior studies have demonstrated association of the oncogenic TCF3-HLF fusion protein with multi-drug resistance via increased expression of ABC and P-glycoprotein drug efflux transporters, as well as via upregulation of pro-survival Ras and BCL-2 pathways. Preclinical studies and small clinical case series of targeted inhibitor addition to chemotherapy or antibody-based and cellular immunotherapies have aimed to improve outcomes for children with TCF3-HLF ALL. Unfortunately, targeting of these activated pathways with the BCL-2 inhibitor venetoclax or other small molecule inhibitors (SMIs) has been minimally or only transiently effective, suggesting more complex mechanism(s) of chemoresistance. In recent years, many patients with relapsed TCF3-HLF ALL have enrolled on clinical trials of CD19- or CD22-targeted immunotherapies. However, TCF3-HLF ALL frequently harbours deactivating mutations in PAX5, a major B-cell regulator and indispensable CD19 transcription factor, placing immunotherapy-treated patients at higher risk of CD19 antigen-loss relapse. New therapies remain needed to prevent relapse and attempt cure. Methods: We designed an unbiased kinome-wide CRISPR/Cas9 library to identify essential drivers in TCF3-HLF leukemogenesis. We screened the human TCF3-HLF ALL cell line HAL-01 and our TCF3-HLF ALL patient-derived xenograft (PDX) model ALL1807 (Hurtz JCI 2020, Schultz Genome Biol 2021), then validated identified targets using 49 SMIs targeting receptor tyrosine kinases (RTK), MEK signaling, and cell cycle pathways. We selected promising candidate inhibitor pairings with non-overlapping mechanisms of action and assessed for in vitro drug synergy via SynergyFinder analyses. Finally, we assessed the in vivo activity of targeted inhibitors in ALL1807 and two newly established TCF3-HLF ALL PDX models (CPCT-0002, CPCT-0003) created from primary pediatric specimens obtained via the LEAP Consortium (Pikman Cancer Disc 2021). Results: RNA-sequencing of HAL-01 and ALL1807 cells followed by functional protein association (STRING) analysis confirmed a network of significantly upregulated (>3-fold) plasma membrane and cytoplasm components of RTK pathways as well as BCL-2. The intersection of the results of the SMI drug library screen with the top 1% targets identified in CRIPSR/Cas9 screen determined p120-RasGAP and Aurora kinase A (AURKA) as therapeutic targets in TCF3-HLF ALL. In vitro treatment of HAL-01 or ALL1807 cells with the RasGAP inhibitor, pluripotin, or the AURKA inhibitor, alisertib, across a range of concentrations demonstrated robust anti-ALL activity. AURKA and RasGAP co-immunoprecipitated and this protein complex was disrupted with alisertib or pluripotin treatment. The AURKB inhibitor barisertib had minimal activity against TCF3-HLF ALL cells, confirming preferential dependency of these cells upon AURKA. Treatment of TCF3-HLF ALL cells with the BCL-2i venetoclax did not disrupt the AURKA/RasGAP complex, suggesting its different mechanism of action and potential for combinatorial drug therapy. Next, we found that alisertib and venetoclax synergistically killed TCF3-HLF ALL cells. Finally, we observed superior inhibition of in vivo leukemia with dual AURKA and BCL-2 inhibitor treatment of three TCF3-HLF ALL PDX models compared to single-agent alisertib or venetoclax (Figure 1). Conclusions: We identified AURKA as a critical new driver in TCF3-HLF ALL via orthogonal genetic and functional assays and confirmed prior observations of BCL-2 dependency in our models. We validated these key targets via in vitro and in vivo pharmacologic inhibition studies with drug synergy detected with combined alisertib and venetoclax in human TCF3-HLF ALL cell lines and PDX models. We posit that dual AURKA and BCL-2 inhibition is a clinically-pragmatic and potentially effective therapeutic strategy for patients with this rare, but highly fatal, leukemia subtype that merits formal clinical investigation. Figure 1 Figure 1. Disclosures Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy. Stegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding. Tasian: Incyte Corporation: Research Funding; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy.

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A Predictive Endothelial-Leukemia Pre-Clinical Platform to Uncover Drug Vulnerabilities for Personalized Treatments

Blood ◽

10.1182/blood-2021-153300 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 704-704

Author(s):

Luca Vincenzo Cappelli ◽

Danilo Fiore ◽

Jude M Phillip ◽

Liron Yoffe ◽

Filomena Di Giacomo ◽

...

Keyword(s):

Clinical Trials ◽

Single Cell ◽

Research Funding ◽

Drug Response ◽

Lymphoblastic Leukemia ◽

Principal Component ◽

Study Drug ◽

Patient Specific

Abstract Background. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few innovative treatment options. This is also contributed by the lack of models capable of capturing the complexity of the tumor and its microenvironment. Aims. To identify patient-specific vulnerabilities and novel therapeutic strategies in T-ALL and interrogate the mechanisms of the crosstalk between leukemic and stromal elements. Methods. We established a drug-testing platform using patient-derived-tumor-xenografts (PDTX) and a mixed-culture approach using E4ORF1-transduced endothelial cells (ECs) (Seandel M et al, PNAS 2008) to overcome host-mediated chemoresistance. We performed functional experiments using total and single-cell RNA sequencing. Results. First, we established a battery of 22 T-ALL PDTX models that matched both phenotypically (immune-histochemistry, flow cytometry) and genotypically (TCR rearrangement, transcriptome) with the primary patients' samples. We then challenged these models (n=14 samples belonging to different PDTX and serial passages within each model) with a library of compounds (n=433) targeting redundant proteins (n=634). Unsupervised clustering and Principal Component Analysis (PCA) demonstrated two clusters of T-ALL samples based on differential drug susceptibility. We could at least partially correlate these differences to specific transcriptomic signatures predictive of drug response (Figure 1A). We then defined a group of pan-active compounds across all models (n=40), which we validated using an independent screening with/without ECs (Figure 1B). We found that ECs counteracted the activity of selected compounds (i.e. TSA, THZ1 and MLN2238). By PCA, we observed distinct response profiles based on different T-ALL models. We vectorized the EC-rescue and found that the direction was the same across all 3 models tested, indicating that it relied on similar mechanisms regardless of model identity. Based on the known role of IGF1-IGFR1 as a supportive EC-rescue axis (Medyouf H et al, J Exp Med 2011), we performed the same screening with/without recombinant IGFBP-7 (500 ng/mL), a decoy IGF1 molecule. Remarkably, IGFBP-7 completely or partially abrogated the EC-mediated rescue of selected compounds [enzastaurin (PKC-β inhibitor), SC144 (GP130 inhibitor), CHIR124 (Chk1 inhibitor) and YM155 (Survivin inhibitor)] (Figure 1B). Drugs not rescued by ECs (n=30) were considered positive hits and 5 of them (ruxolitinib, tofacitinib, panobinostat, bortezomib, irinotecan) ultimately proved to be effective in vivo in randomized pre-clinical trials either alone or in combination (Figure 1C). Our stepwise endothelial-leukemia platform led to the discovery of "public" and "private" vulnerabilities and the proof-of-principle of prediction-guided in vivo pre-clinical trials. We propose a list of compounds that could be readily translated into T-ALL clinical trials (Figure 1D). We finally proved the validity of our platform using other disease models (i.e. B and T-lymphoma PDTXs). Mechanistically, at single-cell resolution, in vitro interacting T-ALL cells and ECs underwent reciprocal transcriptome changes, with T-ALL shifting towards stemness/undifferentiation and ECs towards tumor-ECs (TECs) phenotypes. Furthermore, in vitro EC-educated T-ALL cells mimicked distinct T-ALL subsets of the leukemic spleen of corresponding PDTX mice (Figure 1E). Conclusions. These data demonstrate that our EC-T-ALL culture system simulates in vivo conditions, offering a robust platform to study drug response, leukemia-host interactions and cell plasticity. This approach will improve the pre-clinical predictability of novel drugs/combinations for T-ALL, as well as for other hematologic malignancies, and propel the development of patient-tailored treatments. Figure 1 Figure 1. Disclosures Melnick: Janssen Pharmaceuticals: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Epizyme: Consultancy; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Elemento: AstraZeneca: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Owkin: Consultancy, Other: Current equity holder; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Janssen: Research Funding. Chiaretti: amgen: Consultancy; pfizer: Consultancy; novartis: Consultancy; Incyte: Consultancy. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

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BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

Cancer Cell International ◽

10.1186/s12935-021-01908-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Shuiyan Wu ◽

You Jiang ◽

Yi Hong ◽

Xinran Chu ◽

Zimu Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Lymphoblastic Leukemia ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Rna Seq ◽

Cell Acute Lymphoblastic Leukemia ◽

Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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