Primary T-ALL Research Articles

Abstract 259: Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) patients who experience relapse have a dismal prognosis, with 5-year overall survival rates of 10% for adults and 36% for children. Relapse is attributed to cytotoxic chemotherapy’s inability to target Leukemia Initiating Cells (LICs) that have the potential to re-populate the cancer. There is a need for novel therapeutic strategies to target LIC self-renewal in T-ALL with improved safety profiles and better chances of transition to clinical development. This project utilized a transgenic zebrafish model that accurately recapitulates the most aggressive form of the human T-ALL. This model offered several advantages, including a high frequency of LICs and suitability for drug testing. Over 770 FDA-approved drugs were screened, in vivo using >2,500 syngeneic CG1 zebrafish. Further secondary screening in human T-ALL cells narrowed down the potential hits into 4 compounds that specifically interfere with self-renewal. Finally, the hit drug Amiloride was confirmed by a limiting dilation assay (LDA), resulting in a more than 4-fold decrease in the LIC frequency (p=0.0026). The top hit, Amiloride, which reduced the frequency of LICs in zebrafish and human T-ALL cells, is an inhibitor of the Sodium Hydrogen Exachanger-1 (NHE1). NHE1 has been previously linked to the proliferation, migration, and drug resistance of different cancers. In addition, Amiloride treatment was found to impair mitochondrial function in cancer cells, offering a possible mechanism for the inhibition of LICs. Research has not yet investigated how NHE1, and the altered mitochondrial dynamics related to its inhibition affect the development and persistence of LICs in T-ALL. In our human T-ALL cells, we found that Amiloride significantly reduced mitochondrial basal respiration (p=0.0074), ATP production (p=0.0008) and increased the expression of genes associated with mitochondrial stress such as TFAM, PINK1, PARKIN and MT-CO1. Experiments using shRNA mediated knock down of the NHE1 in human T-ALL cells are underway and will be followed by investigating the impact of NHE1 inhibition on primary patient-derived T-ALL cells in a mouse model. Overall, this project showcases of the power of the zebrafish in cancer research in an efficient and accurate large-scale screen for drugs that impact LIC self-renewal. We have identified Amiloride as a top hit that reduces the frequency of LIC in vivo and in vitro. Finally, we are continuing to investigate the role of the NHE1 in self-renewal in human T-ALL and the associated mitochondrial alterations. Citation Format: Majd Al-Hamaly, Yelena Chernyavskaya, Emma Arvin, Logan Turner, Meghan Haney, Jessica Blackburn. Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 259.

CHD4 Promotes Tumor Formation, Survival, and Resistance to Genotoxic Therapeutics in TP53 Mutant T-Cell Acute Lymphoblastic Leukemia Cells through the NuRD Chromatin Remodeling Complex

T-Cell acute leukemia(T-ALL) though generally responsive to current chemotherapy remains incurable in over 50% of adult patients. Importantly, the survival for patients with TP53 mutant T-ALL disease is less than 10%. We have previously shown the role of the Methyl Binding Domain protein 2-Nucleosome Remodeling and Deacetylase (MBD2-NURD) chromatin remodeling complex in triple negative breast cancer cell growth and the role of the NuRD component, Chromatin-helicase-binding protein 4(CHD4), in tumor colony formation and genotoxic resistance in acute myeloid leukemia. In the present study, we have examined the role of specific NuRD complexes and CHD4 in T-cell acute leukemia cells with mutant TP53. The prototypical NuRD complex possesses both a histone deacetylase (HDCC) sub-complex and an ATPase chromatin remodeling sub-complex containing CHD4 linked to MBD2 or MBD3 of the HDCC. CHD4 is linked to the MBD2/3 anchors of the NuRD complex, via GATAD2A or GATAD2B. Depletion of CHD4 was found to increase the rate of apoptosis in several TP53 mutant T-ALL cell lines including Jurkat, MOLT4, RPMI and CCRF by up to 5 to 10-fold. Likewise, depletion of both MBD2 and MBD3 also increased apoptosis and sensitization to the standard genotoxic chemotherapeutic agent, cytosine arabinoside (Ara-C) but had only minimal effects in normal human CD34+ hematopoietic progenitor cells. Depletion of GATAD2B and GATAD2A by shRNA also resulted in a significantly increased survival of 60 days compared to 20 days in Scramble shRNA controls, in a xenogeneic mouse model of T-ALL. Depletion of CHD4 resulted in a profound (10 to 100-fold) decrease in colony formation of the TP53 mutant T-ALL cell lines in methylcellulose. RNA seq analyses identified the apoptosis, ferroptosis and mitophagy pathways as potential mechanisms for the striking effect of NURD disruption on colony formation. In summary, CHD4, through the NuRD complex, promotes tumor cell formation, survival and therapeutic resistance of TP53 mutant T-ALL cells. Current efforts are aimed at validating these findings in primary T-ALL cells and structure-function studies to advance development of tool compounds for disrupting the NuRD complex, with the long-term goal of developing improved small molecule therapeutics for treatment resistant acute leukemia.

An “Off-the-Shelf” CD2 Universal CAR-T Therapy Combined with a Long-Acting IL-7 for T-Cell Malignancies

INTRODUCTION: T-cell malignancies are associated with frequent relapse and high morbidity in children and adults, partly due to the lack of effective or targeted treatment options. Despite the use of multidrug chemotherapy, <50% of adults and <75% of children with T-ALL survive longer than five years. While CAR-T cell therapy is remarkable in treating B-cell malignancies, its use in T-cell malignancies is restricted due to fratricide, risk of immunodeficiency, and immune escape. METHODS: To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogenic “universal” CD2-targeting CAR-T (UCART2), in which both CD2 and the T-cell receptor alpha subunit are both deleted by CRISPR/Cas9 editing to prevent both fratricide and GvHD. In vitro and in vivo efficacy of UCART2, alone or in combination with rhIL-7-hyFc (efineptakin alfa), a long-acting IL-7, was evaluated using CD2+ HH CBR-GFP T-ALL cell line and primary CD2+ T-ALL PDXs. To determine the impact of CD2 antigen loss on CAR-T cell function, CRISPR-mediated CD2 KO was performed in CD19 targeting CAR-T cells (UCART19ΔCD2). Single-cell secretome analyses on target-stimulated UCART19 and UCART19ΔCD2 were performed using the Isolight platform (IsoPlexis). The in vivo efficacy of UCART19 and UCART19ΔCD2 was evaluated in a CD19+ NALM6 GFP-CBR xenograft model. RESULTS: UCART2 demonstrated efficacy against T-ALL and CTCL in vitro and prolonged the survival of tumor-engrafted NSG mice in vivo. While CD2 deletion in UCART19 had little impact on in vitro killing efficacy, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines, including Granzyme-B and IFN-γ. We also observed that UCART19ΔCD2 (CD2-) had reduced anti-tumor efficacy compared to UCART19 (CD2+) in vivo in a CD19+ NALM6 GFP-CBR xenograft model. Of note, the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc enhanced UCART2 expansion and persistence in vivo(Figure 1A) resulting in increased survival of NSG mice engrafted with both the primary patient T-ALL model (Figure 1B) and in a tumor re-challenge model in vivo. CONCLUSIONS: We have developed an allogenic “universal” CD2-targeting CAR-T cell UCART2, which is effective against T-ALL and CTCL/T-NHL. We found that CD2 deletion in CAR-T cells resulted in reduced secretion of effector cytokines and reduced anti-tumor activity in vivo, indicating that CD2 is critical for CAR-T function. However, when combined with rhIL-7-hyFc (efineptakin alfa), a long-acting recombinant IL-7, UCART2 induced durable complete responses in both primary and tumor rechallenge experiments in vivo. Our data suggest that UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies.

An “off-the-shelf” CD2 universal CAR-T therapy for T-cell malignancies

T-cell malignancies are associated with frequent relapse and high morbidity, which is partly due to the lack of effective or targeted treatment options. To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogeneic “universal” CD2-targeting CAR-T cell (UCART2), in which the CD2 antigen is deleted to prevent fratricide, and the T-cell receptor is removed to prevent GvHD. UCART2 demonstrated efficacy against T-ALL and CTCL and prolonged the survival of tumor-engrafted NSG mice in vivo. To evaluate the impact of CD2 on CAR-T function, we generated CD19 CAR-T cells (UCART19) with or without CD2 deletion, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines (Granzyme-B and IFN-γ). We also observed that UCART19ΔCD2 had reduced anti-tumor efficacy compared to UCART19 in a CD19+NALM6 xenograft model. Of note is that the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc prolonged UCART2 persistence and increased survival in both the tumor re-challenge model and primary patient T-ALL model in vivo. Together, these data suggest that allogeneic fratricide-resistant UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies.

Development of constitutively synergistic nanoformulations to enhance chemosensitivity in T-cell leukemia

Advances in multiagent chemotherapy have led to recent improvements in survival for patients with acute lymphoblastic leukemia (ALL); however, a significant fraction do not respond to frontline chemotherapy or later relapse with recurrent disease, after which long-term survival rates remain low. To develop new, effective treatment options for these patients, we conducted a series of high-throughput combination drug screens to identify chemotherapies that synergize in a lineage-specific manner with MRX-2843, a small molecule dual MERTK and FLT3 kinase inhibitor currently in clinical testing for treatment of relapsed/refractory leukemias and solid tumors. Using experimental and computational approaches, we found that MRX-2843 synergized strongly—and in a ratio-dependent manner—with vincristine to inhibit both B-ALL and T-ALL cell line expansion. Based on these findings, we developed multiagent lipid nanoparticle formulations of these drugs that not only delivered defined drug ratios intracellularly in T-ALL, but also improved anti-leukemia activity following drug encapsulation. Synergistic and additive interactions were recapitulated in primary T-ALL patient samples treated with MRX-2843 and vincristine nanoparticle formulations, suggesting their clinical relevance. Moreover, the nanoparticle formulations reduced disease burden and prolonged survival in an orthotopic murine xenograft model of early thymic precursor T-ALL (ETP-ALL), with both agents contributing to therapeutic activity in a dose-dependent manner. In contrast, nanoparticles containing MRX-2843 alone were ineffective in this model. Thus, MRX-2843 increased the sensitivity of ETP-ALL cells to vincristine in vivo. In this context, the additive particles, containing a higher dose of MRX-2843, provided more effective disease control than the synergistic particles. In contrast, particles containing an even higher, antagonistic ratio of MRX-2843 and vincristine were less effective. Thus, both the drug dose and the ratio-dependent interaction between MRX-2843 and vincristine significantly impacted therapeutic activity in vivo. Together, these findings present a systematic approach to high-throughput combination drug screening and multiagent drug delivery that maximizes the therapeutic potential of combined MRX-2843 and vincristine in T-ALL and describe a novel translational agent that could be used to enhance therapeutic responses to vincristine in patients with T-ALL. This broadly generalizable approach could also be applied to develop other constitutively synergistic combination products for the treatment of cancer and other diseases.

Abstract 423: Induced proximity and proteolytic targeting of LCK as a novel therapeutic approach in T-cell leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer, with a particularly dismal prognosis in patients with relapsed diseases. Our group recently discovered LCK dependency as a therapeutic vulnerability in 33% of T-ALL and LCK inhibitor dasatinib exhibited strong efficacy in vitro and in vivo (Nat Cancer 2, 284, 2021). However, the transient LCK inhibition by dasatinib resulted in only partial responses, and novel agents are needed to suppress LCK signaling in T-ALL in a sustained fashion to achieve long-term efficacy. We synthesized a set of proteolytic targeting chimeras (PROTACs) that bring LCK to cereblon (CRBN) E3 ligase for ubiquitination and degradation. LCK-PROTACs showed extremely high anti-leukemia sensitivity, i.e., up to 1,561.3 fold increase over dasatinib in LCK-activated T-ALL cell line. Complete LCK degradation was induced by most LCK-PROTACs. Based on solubility, permeability, stability, and anti-leukemia effects in vitro, we prioritized SJ001011646 for further evaluations. SJ001011646 induced 50% LCK degradation at the concentration at ~0.001nM, with superior cytotoxic effects in patient-derived primary T-ALL samples compared to dasatinib. In addition, we confirmed the formation of ternary complex of LCK, SJ001011646, and CRBN, using the AlphaLISA assay. In a wash-out experiment, LCK-dependent T-ALL cells were exposed to SJ001011646 or dasatinib for 18 hours and then monitored for viability recovery. Both dasatinib and SJ001011646 led to dramatic growth inhibition within 2 days. While dasatinib-treated cells started to recover 96 hours after drug removal, cells treated with SJ001011646 failed to recover even after 240 hours. To systematically identify therapeutic targets of SJ001011646, we performed proteomic profiling of T-ALL cells before and after drug treatment in vitro. Overall, 126,670 unique peptides were identified and mapped to 10,158 proteins, of which LCK was most significantly reduced by PROTAC. In parallel, kinome-binding profiling confirmed LCK as a primary target of SJ001011646. Finally, we explored pharmacokinetic and pharmacodynamic properties of SJ001011646 in vivo, using patient-derived xenograft models of T-ALL. Given at the same dosage level as a single injection, SJ001011646 showed 367% increase in the duration of LCK suppression compared to dasatinib. Taken together, we described the development and preclinical evaluation of LCK-targeting PROTACs in T-ALL. Inducing proximity to CRBN and therefore degradation, these agents can produce sustained LCK suppression with much improved anti-leukemia efficacy than small molecule inhibitors, and are therefore promising novel therapeutic agents in T-ALL. Citation Format: Jianzhong Hu, Jamie Jarusiewicz, Jaeki Min, Lei Yang, Divyabharathi Chepyala, Marisa Actis, Guoqing Du, Brandon Smart, Dylan Maxwell, Boer Xie, Satoshi Yoshimura, Junming Peng, David T. Teachey, Zoran Rankovic, Jun J. Yang. Induced proximity and proteolytic targeting of LCK as a novel therapeutic approach in T-cell leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 423.

Abstract SY04-03: Identification of SIRT1 as a novel therapeutic target in T-ALL

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of TALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstrated the importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in TALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemias leads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. Citation Format: Daniel Herranz. Identification of SIRT1 as a novel therapeutic target in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY04-03.

Novel in Vitro and In Vivo Drug Screening Platform for ALL.

Acute Lymphoblastic leukemia (ALL) is a malignancy of bone marrow. Accumulation of mutations in lymphoid progenitor cells give rise to either B-ALL or T-ALL. Treatment for ALL has improved in recent years, yet relapse of the disease and development of resistance are observed in patients. Lack of suitable and robust in vitro and in vivo drug testing platforms for primary ALL cells along with the lack of rapid development of novel therapeutics drugs encompassing the heterogeneity of the disease contribute to the delay in approved patient treatments.We have developed a short-term culture system that supports the survival of primary B-ALL and T-ALL cells. Our ALL bank includes patient-derived specimens with complete cytogenetics and surface marker expression information. Different culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL and T-ALL specimens. Cell growth/viability was assessed using the Cell Titer-Glo ® assay. Primary B-ALL cells survived in the optimized media for 3 days and a heterogenous dose dependent response was observed across the models to chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. BCR-ABl - B-ALL patient samples were found to be resistant to imatinib in contrast to BCR-ABL + samples which were sensitive to imatinib. Similarly, culture conditions optimized for T-ALL primary cells supported the survival until day 6 and displayed a diverse response to standard of care drugs like venetoclax, imatinib, vincristine, cytarabine and methotrexate, reflecting the heterogeneity of the patient derived specimens. Immunophenotypic characterization of ALL cells grown in culture displayed retention of the B and T cells surface marker expression.Further, a patient derived pre-clinical xenograft model was developed in NCG mice to study in vivo ALL drug efficacy. 100% engraftment was observed for B-ALL primary cells, with latency of engraftment (>3%) in peripheral blood varying from 15 days to 3.5 months. 30-90% of the bone marrow cells were occupied by human CD45 cells. Infiltration of human B-ALL cells were observed in the spleen causing splenomegaly. 8 out of the 14 models having high penetrance were passaged until P3. Flow analysis at each passage demonstrated surface marker expression displaying low divergence from the primary samples. Additionally, evaluation of ex vivo drug response from B-ALL PDX splenocytes was largely concordant with the primary specimen ex vivo data in three of the models evaluated. In an in vivo drug efficacy study administration of venetoclax, CHOP and R-CHOP inhibited the proliferation of B-ALL cells. Significant reduction of B-ALL cells was observed while on treatment with Venetoclax. At termination of the study, up to 80% reduction of human B-ALL cells was observed in whole blood, bone marrow, and spleen after treatment with CHOP and R-CHOP in comparison to the vehicle cohort.Similarly, patient derived T-ALL pre-clinical xenograft model development is in progress. Thus, we have developed a robust in vitro drug testing platform for B-ALL and T-ALL to evaluate drug efficacy. We also demonstrate that NCG mice support the growth and proliferation of primary B-ALL cells and have successfully developed an in vivo platform that will facilitate the testing of clinically relevant chemotherapeutic drugs for ALL. DisclosuresNo relevant conflicts of interest to declare.

Anti-CD21 Chimeric Antigen Receptor (CAR)-T Cells for T Cell Acute Lymphoblastic Leukaemia (T-ALL)

CAR-T cell therapy against CD19 has changed the treatment landscape in relapsed/refractory (r/r) B-ALL. R/r T-ALL has a dismal prognosis, with an unmet need for effective targeted therapies. Several unique challenges mean that CAR-T cell therapy has yet to be successfully translated to T-ALL. Most strategies have targeted pan-T cell antigens (CD7, CD5) but these are limited by T cell aplasia and fratricide, requiring elimination of CAR-T antigen surface expression during manufacture. An ideal target would be exclusively or largely confined to the malignant T cell component but published examples of these (CD1a and TRBC1) are expressed in only minor T-ALL subsets.We previously showed that CD21 is expressed in a NOTCH-dependent manner in T-ALL (Leukemia. 2013, 27:650) and have developed it as a potential immunotherapy target, being primarily expressed on normal B cells, with minimal expression on mature T cells.70% of human T-ALL cell lines (9/16) expressed surface CD21 by flow cytometry (FACS), with a median antigen density in positive lines of 2545/cell. In primary T-ALL, 57% of presentation samples (n=58) expressed CD21 (median antigen density 1168/cell). 45% of relapse (n=11) and 20% of primary refractory cases (n=30) expressed CD21, with a similar antigen density to presentation samples. CD21 positivity varied by maturational stage, with highest expression in cortical T-ALL (80% of cases) followed by pre-T (72%), mature (67%), ETP (25%) and pro-T (17%).Healthy donor blood (n=14) showed CD21 expression limited to B cells and a low proportion (11%) of T cells (10-fold lower intensity v B cells, 316 antigens/cell). T cell CD21 expression was not up-regulated upon activation with CD3/CD28 antibodies (n=6) and was not associated with markers of differentiation/exhaustion.To target CD21, DNA gene-gun vaccination of rats with a plasmid encoding full-length CD21, followed by phage display was performed and multiple anti-CD21 scFvs isolated. These were cloned into 4-1BBζ CARs and expressed in primary T cells but failed to kill or secrete cytokines in response to CD21+ SupT1 cells.CD21 is a bulky molecule, with 15/16 sushi repeats in the extracellular domain. All isolated scFvs were found to bind membrane-distal domains. We hypothesized that ineffective signalling due to inadequate synapse formation was responsible for poor performance of anti-CD21 CAR-T, and that binders to membrane-proximal epitopes would signal more efficiently. We re-vaccinated rats with the first 5 sushi repeats of CD21 and generated a library of binders which bound CD21 at this membrane-proximal region.Multiple candidate binders expressed as CARs were functional, with cytotoxicity and interferon-γ secretion in response to CD21+ target cells. However, non-specific background cytokine secretion was seen against CD21 negative cells, and no IL-2 secretion was seen. Re-cloning binders into a fragment antigen binding (Fab)-CAR architecture yielded constructs capable of specific cytotoxicity, IFN-γ and IL2 secretion against a CD21+ cell line but not its CD21 negative counterpart (n=6). Our lead anti-CD21 candidate CAR specifically proliferated in vitro, without fratricide or premature exhaustion/ differentiation, and was active against low-density CD21-positive cell lines (n=3) and primary cells from 2 T-ALL patients. Improved functionality of Fab v scFv-based CAR was not driven by higher affinity binding or CAR surface expression.We tested anti-CD21 CAR in murine models of T-ALL. NSG mice were injected with SupT1-luciferase cells and treated with aCD19 or aCD21 CAR-T on day +5. At 2 weeks post treatment, markedly lower disease burden was seen in CD21 CAR-T v CD19 recipients by bioluminescence imaging (median radiance 71700 v 790000 p=0.0079). Further, we injected primary T-ALL blasts in another cohort, treating with aCD19 or aCD21 CAR-T on D+20. Serial bleeds from day 27 post CAR-T showed tumour control in aCD21 CAR treated mice (p=0.024) with an overall survival advantage (median OS 44 days vs undefined, HR = 19.8, p = 0.0069, n=4/group).In summary, we propose CD21 as a novel target for CAR-T cell therapy in T-ALL. Its expression is largely restricted to the malignant T cell compartment, overcoming issues with fratricide and on-target off-tumour effects seen in many T-ALL CAR-T strategies to date. Despite the complexity of the target, we have successfully generated an aCD21 CAR that is functional both in vitro and in vivo. DisclosuresMaciocia: Autolus: Current equity holder in publicly-traded company. Onuoha: Autolus: Ended employment in the past 24 months. Khwaja: Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Maciocia: Autolus: Current equity holder in publicly-traded company, Research Funding. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company.

Maturity State and MCL-1 Dependence Predetermines Response to NOTCH1 Inhibition in T-ALL

Introduction:Acute T cell lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and young adults that frequently becomes treatment-refractory and relapses. The Notch1 pathway is a key oncogenic driver in T-ALL and is aberrantly activated in more than 50% of the cases. Despite promising pre-clinical data using gamma secretase inhibitors such as DBZ to target NOTCH1, resistance is rapidly occurring in vivo. As molecular heterogeneity has been linked to treatment escape, we focused our study on defining transcriptional cell states driving resistance to NOTCH inhibition and understanding their relation to mitochondrial priming.Methods:5 primary T-ALLs harboring NOTCH activating mutations were engrafted in NSG (NOD-scidIL2Rgnull) mice. Upon reaching ~ 10% of human CD45+ positive leukemic blasts in the peripheral blood, randomized groups of 8 mice per primary T-ALL were treated with DBZ (Dipenzazepine; 10 μM/kg every other day through tail vein) or vehicle (VEH). 3 mice per group were sacrificed after one week of treatment to assess short-term effect of DBZ, while the remaining 5 mice were weekly monitored for disease progression, leukemic blasts were collected from lymphoid organs and overall survival was determined.Full-length transcriptome analysis of 3188 blasts present in the blood of 20 sensitive and 22 refractory mice was performed by Smart-Seq2. Based on scRNA features, ‘scVelo’ and ‘CytoTRACE’ were used to identify developmental potential and differentiation trajectories. Cell fate and transcriptional regulatory networks were defined and reconstructed using ‘SCENIC’.Assessment of mitochondrial priming as measured by BH3 profiling was used to identify anti-apoptotic vulnerabilities present in these PDX models.Results:Upon DBZ, short or long-term disease control was observed in two strains, while rapid resistance occurred in three strains, thus establishing two sensitive and three refractories to NOTCH inhibition PDX models. Immunohistochemical analysis showed decreased expression of active NOTCH1 in spleen biopsies of all strains, validating the efficacy of DBZ and suggesting a mechanism of resistance independent of ICN1.Single cell transcriptional profiling showed enrichment of immature hematopoietic signatures and co-expression of lymphoid and myeloid progenitor programs in refractory models. Interestingly, pre-existing cells harboring refractory-like transcriptional circuits within the untreated sensitive population were identified. Upon treatment, despite increased differentiation in all models, lineage promiscuity was maintained in refractory strains, suggesting that cellular plasticity mediates treatment escape.Next, we characterized cell states driving treatment refraction. RNA velocity projections identified two distinct immature states differing in cell cycle and oncogenic signaling. Clustering of untreated, sensitive leukemic cells in immature state imply that aberrant lineage commitment can predict response to NOTCH inhibition in vivo. These observations were further confirmed by differentiation state analysis, where prior to treatment, high developmental potential was correlated to treatment escape. Surprisingly, in addition to early lineage differentiation drivers such as BCL11A, state-specific regulons analysis associated immature states with BCLAF1 a transcriptional regulator of apoptosis.We postulated that these transcriptional circuits lead to differential apoptotic priming, therefore the dependence on individual anti-apoptotic proteins was evaluated. Mitochondrial priming at baseline revealed BCL-2 dependence in sensitive strains whereas MCL1-dependence was observed in refractory ones. Upon DBZ treatment, while dependency profiles in refractory strains remained unchanged, a functional switch from BCL-2 to MCL1-dependency occurred in sensitive models.Conclusion:Our results suggest that response to NOTCH inhibition is predetermined by cell maturity states and their associated transcriptional circuits responsible for differential sensitivity to apoptotic priming via BCL2 and MCL1. These data suggest that combining BH3 and lineage commitment profiling may predict drug responses in vivo. Moreover, our findings highlight the importance of targeting co-existing cell states to overcome transcriptional heterogeneity as a driver of treatment escape. DisclosuresLetai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Weinstock: Daiichi Sankyo: Consultancy, Research Funding; Verastem: Research Funding; Abcuro: Research Funding; Bantam: Consultancy; ASELL: Consultancy; SecuraBio: Consultancy; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

Therapeutic Targeting of Mertk and BCL-2 in T-Cell and Early T-Precursor Acute Lymphoblastic Leukemia

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL and is associated with inferior outcomes relative to B-cell ALL. Early T-precursor ALL (ETP-ALL) is a subset of T-ALL characterized by an immature T cell phenotype, resistance to therapy, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in 40-50% of T-ALLs, particularly those with an immature T cell phenotype, suggesting a role in ETP-ALL. Inhibition of MERTK using shRNA delayed leukemia progression and prolonged survival in a T-ALL xenograft model, implicating MERTK as a therapeutic target. MRX-2843 is an orally available dual MERTK/FLT3 inhibitor currently in phase I clinical trials. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is specifically expressed in immature T cell precursors, is preferentially expressed in ETP-ALL compared to other T-ALLs, is essential for ETP-ALL cell survival, and is regulated downstream of MERTK in acute leukemia cells. Thus, combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL.MethodsLoucy and PEER ETP-ALL cell lines were cultured with vehicle or MRX-2843. Phosphorylated and total MERTK were assessed by immunoblot. Relative cell numbers were measured using Presto Blue reagent. Cells were stained with PoPro-1-iodide and propidium iodide and apoptotic and dead cells were quantitated by flow cytometry. T-ALL patient samples were cultured with UNC2025, a close analogue of MRX-2843, and relative cell numbers were assessed using MTS reagent. Orthotopic xenografts were established in NSG or NSGS mice using luciferase-expressing Jurkat cells (T-ALL), luciferase-expressing Loucy cells (ETP-ALL) or an ETP-ALL patient sample and leukemia burden was monitored by bioluminescence imaging or flow cytometry. MRX-2843 (65 mg/kg or 75 mg/kg) or saline vehicle were administered orally once daily. Differences in disease burden were assessed with the Mann-Whitney-U test or one-way ANOVA. Survival was determined by Kaplan-Meier analysis. Loucy and PEER cells were plated and screened in quadruplicate against >150 pairwise combinations of MRX-2843 and the BCL-2 inhibitor venetoclax in a high-throughput format. Synergy was calculated using the response additivity model.ResultsTreatment with MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK, inhibited expansion of ETP-ALL cells, and induced cell death in vitro. Fifty-four percent (21/39) of primary T-ALL patient samples were sensitive to UNC2025 with an IC 50≤550 nM, including 2/5 (40%) pediatric samples and 10/19 (53%) adolescent/young adult samples. Treatment with MRX-2843 significantly reduced leukemia burden in cell line-derived T-ALL and ETP-ALL xenograft models and prolonged survival by 50% and 13% in the T-ALL (n=10, p<0.0001) and ETP-ALL (n=10, p=0.0136) models, respectively. Similarly, in a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 reduced peripheral blood disease burden by 83% and spleen weight by 64% compared to vehicle-treated mice (n=8, p<0.001) and prolonged survival by 41% (n=8, p=0.0016). MRX-2843 mediated anti-leukemia activity in combination with venetoclax and a dose ratio of 1:20 MRX-2843:venetoclax provided optimal synergy in Loucy and PEER ETP-ALL cells in vitro (Figure 1).ConclusionsMRX-2843 has therapeutic activity in ETP-ALL cell culture and xenograft models and over half of T-ALL patient samples were sensitive to MERTK/FLT3 inhibition. MRX-2843 also mediated synergistic anti-leukemia activity against ETP-ALL cells in combination with venetoclax, with an optimal molar ratio of 1:20. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL, suggest that MRX-2843 may be particularly active alone and in combination with venetoclax in the ETP-ALL subset, and provide rationale for clinical testing of MRX-2843, with the ultimate goal to progress to trials evaluating MRX-2843 in combination with other agents. Toward this end, MRX-2843 monotherapy will be tested in patients with relapsed leukemia in an upcoming clinical trial (NCT04872478). [Display omitted] DisclosuresWang: Meryx: Other: Equity ownership; University of North Carolina: Patents & Royalties. Frye: University of North Carolina: Patents & Royalties; Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Earp: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Tyner: Petra: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Janssen: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Constellation: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Agios: Research Funding. DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Abstract LB147: Updates on clinical safety and efficacy result of GC027, the first-in-human, “Off-the-Shelf” CD7 CAR-T stand-alone therapy for adult patients with relapsed/refractory T-cell lymphoblastic leukemia (r/r T-ALL)

Abstract Introduction: T-ALL represents an area of high unmet medical need. Once relapsed, patients have limited treatment options. We first reported data from a single-arm, open-label, multi-center, investigator-initiated study in adults with r/r T-ALL (ChiCTR1900025311) treated with TruUCAR™ GC027, an off-the-shelf CAR-T product at AACR 2020. Here we report long term follow-up results and preliminary results of additional patients treated. Methods: Between July 2019 to Feb 2021, a total of 6 r/r T-ALL patients (19-38 yrs) with a median of 6 prior lines were treated with a single infusion of GC027 at 3 different dose levels after Lymphodepletion over 6 days: DL1 (6x106 cells/kg, n=2), DL2 (1x107 cells/kg, n=3), DL3 (1.5x107 cells/kg, n=1) including a primary refractory T-ALL pt with extramedullary (EM) disease. Results: All 6 pts treated showed molecular expansions of CAR-T cells and peaked day 6-11 in peripheral blood (by qPCR, median 416,905 copies/ug DNA). Robust cellular expansion (by FACS) was observed in 5 pts (median 648 cells/ul). Clinically, 5/6 pts achieved MRD-CR/CRi at the month 1 assessment, including 1 pt with EM disease at several locations including neck, axilla, mediastinum and pancreas. His EM lesions were reassessed by PET-Scan on day 47 after treatment, confirming a complete response. Long-term follow-up (n=5): All pts evaluable at day 28 (n=4) reconstituted all blood cell lineages as assessed by blood cell count. At 6 months, 3/5 pts had maintained MRD- CR. At data cut-off Feb. 4, 2021, 1 pt continued to be in MRD-CR at 16.8 months (day 505) post treatment. 1 pt maintained CR until day 267, when he was found CD7 partial negative (by FACS) relapsed (BM). 1 pt with primary refractory disease (no response to VDP) maintained MRD- CR until month 7 (day 217) and was found relapsed by BM and PB assessment with CD7 partial negative (FACS). He received HSCT on day 255, achieved MRD- CR but relapsed again 5 months post HSCT. Safety (n=6): Overall safety findings were consistent with previous observations reported. TEAEs occurred were grade 3 febrile neutropenia (6/6), grade 4 neutropenia (4/6), grade 4 thrombocytopenia (4/6) and grade 3 anemia (3/6). All TEAE resolved after treatment with SOC and best supportive care, neutropenia improved with G-CSF treatment. Non-hematological TEAE presented as grade≤2 hypoalbuminemia (6/6), grade 3 pulmonary infection (3/6), grade≤2 AST increase (4/6) and grade≤2 ALT increase (3/6). 6/6 pts developed grade ≥ 3 CRS (ASBMT consensus grading), which was manageable in all cases and resolved after treatment. No Grade 5, ICANs or aGvHD were observed. Conclusion: The allogeneic off-the-shelf TruUCAR™ CAR-T GC027 shows promising efficacy as standalone therapy in heavily pretreated R/R T-ALL pts with a manageable safety profile. A single infusion of GC027 was able to induce deep, durable responses, with the longest duration of response (MRD-CR) 18 months post treatment still ongoing without any additional therapy during remission. GC027 warrants further evaluation in larger clinical studies. Citation Format: Shiqi Li, Lei Gao, Zhongtao Yuan, Kun Wu, Lin Liu, Le Luo, Yao Liu, Cheng Zhang, Yu Li, Duanpeng Wang, Zhimin Li, Xu Tan, Ruihao Huang, Dingsong Zhang, Youcheng Wang, Xinxin Wang, Jia Liu, Martina Sersch, Sanbin Wang, Xi Zhang. Updates on clinical safety and efficacy result of GC027, the first-in-human, “Off-the-Shelf” CD7 CAR-T stand-alone therapy for adult patients with relapsed/refractory T-cell lymphoblastic leukemia (r/r T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB147.

Abstract 3185: Integrin signaling is critical for myeloid-mediated support of T-ALL

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a malignant transformation of immature T cells. Although T-ALL harbors multiple pro-leukemic genetic alterations, T-ALL blasts require exogenous signals to survive. We recently reported that tumor-associated myeloid cells provide critical support for T-ALL in mouse models and in pediatric patient samples, at least partially through activation of IGF1R signaling. However, IGF1 alone is not sufficient to support T-ALL survival, implicating additional mechanisms in myeloid-mediated T-ALL support. We previously found that T-ALL cells make prolonged and frequent contacts with tumor-associated myeloid cells in live tissue sections; thus, we hypothesized that cell-cell interactions are essential for myeloid-mediated support of T-ALL. Here, we confirm that cell contact is required for myeloid cells to support T-ALL cell survival in vitro. Analysis of transcriptional profiling data revealed enrichment of gene signatures associated with integrin pathways in T-ALL cells relative to healthy T cells, implicating integrin-mediated cell adhesion as a potential mechanism underlying contact-dependent T-ALL support. Blockade of integrin αL ± integrin β1 or their ligands ICAM-1 ± VCAM-1 via antibodies or pharmacologic inhibitors significantly decreased T-ALL survival and reduced activation of IGF1R in T-ALL cells co-cultured with tumor-associated myeloid cells. Consistent with these findings, inhibition of integrins or their ligands in vivo via antibody blockade or genetic manipulation resulted in a significant reduction of T-ALL burden at multiple leukemic sites and prolonged survival. Consistent with mouse models, blockade of integrin-mediated cell adhesion significantly reduced survival of primary pediatric T-ALL cells co-cultured with myeloid cells. Furthermore, analyses of published transcriptomic datasets from pediatric T-ALL patients showed that enrichment of integrin pathways correlates with elevated myeloid gene signatures and poor patient prognosis. Together, our studies demonstrate that tumor-associated myeloid cells promote survival of T-ALL cells by activating integrin signaling, implicating the associated integrins and downstream signals as targets for therapeutic intervention. Citation Format: Aram Lyu, Seo Hee Nam, Zicheng Hu, Dhivya Arasappan, Terzah M. Horton, Lauren I. Ehrlich. Integrin signaling is critical for myeloid-mediated support of T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3185.

The Crucial Role of ROS-HO-1 Dependent Apoptosis Induced By Pan-HDACi in T-Cell Acute Lymphoblastic Leukemia

Histone hypoacetylation occurs in many hematological malignances and histone deacetylase inhibitors (HDACi) is a promising approach to treat hematological malignances. The purpose of our study was to further investigate the mechanisms and efficiency of pan-HDAC inhibition (AR-42) in primary human TALL cells and T-ALL cell line. Our previous study demonstrated significantly increased mRNA and protein expression of HDAC1 HDAC2 HDAC3 HDAC7 and HO-1 in T-ALL patients(p <0.01); the expression of HDAC1and HDAC2 and HO-1 were associated with the risk stratification. Furthermore HO-1 overexpression was mediated by constitutively activated NF-κB, which were confirmed by EMSA, immunofluorescence and immunoblotting. In present study, primary cell of 25 T-ALL patients and Jurkat were treated with AR-42, and cell viability, apoptosis, cell cycle, mitochondrial membrane potential(MMP) and effects on signaling pathways were examined. Our data showed AR-42 was cytotoxic to T-ALL cells with IC50 values of 0.325±0.082 and 0.296±0.064μM for T-ALL primary cell and Jurkat; AR-42 arrested Jurkat cells at G0/G1 phase with the increasing expression of p21; AR-42 induced apoptosis with cleavage of caspases3,8 and 9, and cell apoptosis was largely prevented by caspase inhibition (Z-VAD-FMK). AR-42 induced apoptosis through an increase in the ROS production and the disruption of mitochondrial membrane potential, and cell apoptosis induced by AR-42 was significantly diminished by ROS scavengers NAC (N-acetylcysteine). AR-42 downregulated the expression of HO-1 and inhibited activation of NF-κB. Overexpression of HO-1 by a lentivirus construct partly prevent apoptosis induced by AR-42. Together, these data demonstrate that AR-42 can induce caspase-dependent apoptosis mainly through ROS-(NF-κB)- (HO-1) pathway in T-cell acute lymphoblastic leukemia. DisclosuresNo relevant conflicts of interest to declare.

Repression of the Orphan Nuclear Receptor Esrrb Results in Glucocorticoid Resistance in ALL

▪Acute lymphoblastic leukemia (ALL) is an aggressive hematological malignancy, in which 20-30% of pediatric patients relapse or undergo induction failure (Vrooman, 2009). ALL is treated with a combination of chemotherapeutic agents, including glucocorticoids (GCs). Pediatric ALL patients who exhibit resistance to GCs at diagnosis or relapse have a poor prognosis(Dördelmann, 1999). We performed an unbiased whole genome survival-based shRNA screen in primary mouse T-ALL cells to identify GC resistance genes. This shRNA screen identified known mediators of GC resistance as well as novel genes involved in chromatin remodeling, cell differentiation and metabolism.One of the top hits in the GC resistance screen were shRNAs targeting the orphan nuclear receptor ESRRB. ESRRB is essential for the maintenance of pluripotency in mouse embryonic stem cells and decreases in ESRRB expression stimulate differentiation (Zhang, 2008). We observe no effects of ESRRB deficiency on leukemic cell growth or differentiation. However, silencing of Esrrb prevents GC-induced apoptosis in mouse and human T-ALL cell lines in vitro . Although the screen was done in mouse T-ALL cells, we find that ESRRB-deficiency also results in GC resistance in human B-ALL cells. As many published GC resistance genes fail to translate in vivo, we transplanted mice with T-ALL cells transduced with shRNAs targeting Esrrb or empty vector. Dexamethasone treatment prevented disease progression in mice transplanted with control T-ALL cells however, dexamethasone treatment had no significant effect on disease development in mice transplanted with ESRRB deficient T-ALL cells, indicating that ESRRB deficiency confers GC resistance in vivo.As ESRRB has no established role in T-cells or the GC response, we performed RNA-sequencing on normal and ESRRB-deficient T-ALL cells in the absence and presence of GCs to uncover the mechanism of GC resistance. ESRRB deficient T-ALL cells fail to upregulate 65% of GC-induced genes, indicating that decreases in ESRRB expression prevents optimal GC-induced gene expression and apoptosis. Furthermore, we identified Esrrb as a GC-induced gene, supporting its role in the GC response. Due to the global repression of GC-induced gene expression in ESRRB-deficient leukemic cells, we examined GC receptor (GR) localization, but found that ESRRB had no detectable effects on GR localization.Collectively these data suggest that ESRRB may function as a novel co-activator of GR mediated transcription. Consistently, we found ESRRB expression decreased in a subset of relapse ALL patients, suggesting that ESRRB may contribute to GC resistance in patients. These findings identify ESRRB as a novel GR co-activator, whose expression is suppressed in a subset of relapse ALL patients. We demonstrate that treatment with the synthetic ESRRB agonist GSK4716 stimulates GC-induced apoptosis and gene expression in human ALL cell lines. These data raise the intriguing possibility that ESRRB agonists may re-sensitize GC resistant leukemia patients to GC therapy. DisclosuresNo relevant conflicts of interest to declare.

PDX Models Recapitulate Genetics and Epigenetics of Pediatric T-Cell Leukemia

Patient-derived xenografts (PDX) emerged as important tools to study leukemia biology and potential therapeutic regimens. Here, we compared cellular, molecular and developmental features of PDX-derived blasts with primary pediatric precursor T-cell acute leukemia (T-ALL) cells. The cells were obtained at the times of initial diagnosis and relapse. We analyzed the samples at the genomic level (whole genome sequencing (WGS), whole exome sequencing (WES), multiplex ligation probe amplification (MLPA), targeted sequencing) and the epigenetic level (DNA methylation, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq)).Altogether 75% of our samples engrafted successfully. Of these 12 were matched pairs of initial diagnosis and relapse as well as corresponding PDX. Of further 12 matched pairs either initial diagnosis (2/12) or relapse (7/12) or neither (3/12) engrafted indicating that relapse samples tend to engraft more successfully (p= 0.1824 (McNemar's test)), which is in agreement with observations in other cancer entities.In 11 out of 12 matched pairs (initial and relapse) of primary and PDX genomic stability of the PDXs was assessed using WGS and WES. Ninety-two % of the mutations with an allele frequencies (AF) >30% were conserved. Of altogether 609 SNVs and InDels (AF>10%) detected in primary samples 68 % (417) were also detected in the corresponding PDXs. In some PDXs, mutations not identified by targeted sequencing in the primary sample (NOTCH1, PHF6, DNA11, USP9X) occured at a high AF (22-59%), which may have been the result of selection of a subclone or of an acquisition of the mutation in the process of PDX generation.Thirty of 35 (86%) large CNA assessed by WGS were preserved in corresponding PDX models indicating that CNAs are likely earlier events in the leukemogenesis that are present at high AF in the primary samples. CNA analysis with MLPA confirmed that PDXs retained 96% (80/83) of the deletions and amplifications of primary leukemia.In 18 out of the matched 22 PDXs all clonal mutations (AF >30%) detected in the primary sample have been preserved in the PDX. Seventeen out of 22 contained more than 1 clone. Fifteen out of these 17 retained the complexity of primary sample with different subclones successfully engrafted.When considering the 11 matched pairs, we observed 4 different engraftment types of initial disease. Type 1 engrafted in a similar way in which they relapsed preserving all or nearly all mutations found in patients' sample (4 samples). Type 2: the relapsing clone that was not detected in initial diagnosis was present in PDX derived of initial diagnosis (PDX_INI) but it was not dominant (3). Type 3: the relapsing clone that was present at initial diagnosis did not engraft into PDX_INI (2). Type 4 preserved the relapsing clone as well as the mutations specific for initial diagnosis (2). Out of 11 PDXs of relapse 9 preserved all or nearly all mutations detected in primary sample. In 2/11 PDXs of relapse one of the clones preexisting in primary relapse was selected for, while the other one was lost.Differential methylation analysis by EPIC array of 21,711 promoters of primary samples and PDXs revealed that the average methylation in primary samples (β=0.43) did not differ from that in PDXs (β=0.45). Principal Component Analysis and unsupervised hierarchical clustering showed that matched pairs, both of the primary and the PDX samples cluster together (ρ=0.9912). Similarly, analysis of chromatin accessibility by ATAC-seq showed that more than 92% of peaks of a height > 20 and more than 75% of the peaks of a height of > 10 were preserved between primary samples and corresponding PDXs.In conclusion, PDX models largely reflect the heterogeneity and the complexity of the primary leukemic sample at the genomic and epigenomic level, although some clonal and particularly subclonal mutations can be lost. Differences in engraftment and various engraftment scenarios of initial diagnosis in contrary to relapse samples suggest that relapse samples are more preselected and stable in engraftment. Altogether, this overall genomic and epigenomic stability of the primary T-ALL in the PDX renders the models as a suitable source for material when primary samples are limiting and also to reduce uncontrollable pre-analytical changes in primary samples. Further, these models are suitable for the preclinical development of new treatment strategies for pediatric T-ALL. DisclosuresSchrappe:Baxalta: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding.

Base-edited CAR T cells for combinational therapy against T cell malignancies

Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

A novel and highly effective mitochondrial uncoupling drug in T-cell leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted adenosine monophosphate–activated serine/threonine protein kinase (AMPK) activation and downregulation of mammalian target of rapamycin (mTOR) signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL patient-derived xenografts (PDXs) led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47–driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease.

Chimeric Antigen Receptor T Cells Targeting NKG2D-Ligands Show Robust Efficacy Against Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia.

CAR T cell approaches to effectively target AML and T-ALL without off-tumor effects on healthy myeloid or T cell compartments respectively are an unmet medical need. NKG2D-ligands are a promising target given their absence on healthy cells and surface expression in a wide range of malignancies. NKG2D-ligand expression has been reported in a substantial group of patients with AML along with evidence for prognostic significance. However, reports regarding the prevalence and density of NKG2D-ligand expression in AML vary and detailed studies to define whether low level expression is sufficient to trigger NKG2D-ligand directed CART cell responses are lacking. NKG2D ligand expression in T-ALL has not previously been interrogated. Here we report that NKG2D-ligands are expressed in T-ALL cell lines and primary T-ALL. We confirm that NKG2D-ligands are frequently surface expressed in primary AML, albeit at relatively low levels. Utilizing CAR T cells incorporating the natural immune receptor NKG2D as the antigen binding domain, we demonstrate striking in vitro activity of CAR T cells targeting NKG2D-ligands against AML and T-ALL cell lines and show that even low-level ligand expression in primary AML targets results in robust NKG2D-CAR activity. We found that NKG2D-ligand expression can be selectively enhanced in low-expressing AML cell lines and primary AML blasts via pharmacologic HDAC inhibition. Such pharmacologic NKG2D-ligand induction results in enhanced NKG2D-CAR anti-leukemic activity without affecting healthy PBMC, thereby providing rationale for the combination of HDAC-inhibitors with NKG2D-CAR T cell therapy as a potential strategy to achieve clinical NKG2D-CAR T cell efficacy in AML.

CRISPR-Cas9 Knock out of CD5 Enhances the Anti-Tumor Activity of Chimeric Antigen Receptor T Cells

CRISPR-Cas9 Knock out of CD5 Enhances the Anti-Tumor Activity of Chimeric Antigen Receptor T Cells