Primary T-cell Acute Lymphoblastic Leukemia Research Articles

Significance of immunophenotypic, cytogenetic, and molecular markers in adult patients with T-cell lymphoblastic leukemia

Background. Current chemotherapy protocols for T-cell acute lymphoblastic leukemia (T-ALL) allow achieving a 5-year overall survival of 60–90 %, but relapsed and refractory forms remain incurable situations.Aim. To determine the significance of immunophenotypic, cytogenetic and molecular markers in adult T-ALL patients receiving therapy according to the ALL-2016 protocol.Materials and methods. From December 2016 to June 2022, 113 patients with primary T-ALL were included in the study. Cytogenetic study was performed in 104 (92 %) patients; anomalies in the IKZF1 and NOTCH1 genes were investigated in 43 (38 %) patients.Results. The worst prognosis was in patients with ETP and near-ETP variants of T-ALL (3-year disease-free survival was 54 % in ETP group, 33 % in near-ETP group vs TI/II – 79 %, TIII – 89 %, TIV – 75 %). In early T-ALL variants, abnormal karyotype was most common (ETP – 80.7 %, near-ETP – 60 %). Aberrations in NOTCH1 gene were found in 53 % of cases (in 23 out of 43 patients), and no mutations were found in IKZF1 gene in our study. In the group with no NOTCH1 abnormalities, the overall survival was significantly worse than in the group with abnormalities (NOTCH1– – 52 % vs NOTCH1+ –81 %; p = 0.05).

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.

CD28 CAR T Cells for the Treatment of T Cell Malignancies

CONCLUSION While synthetic immunotherapy has been introduced into the standard of care treatment of patients with B-lineage malignancies, patients with T-lineage malignancies have not gained benefit from this novel approach, yet. The main reason is the challenging choice of target antigen in T-lineage malignancies such as pediatric T cell acute lymphoblastic leukemia (T-ALL). As activating mutations of CD28 have already been described in T cell malignancies, we set out to quantify surface expression of CD28 and other costimulatory molecules on primary pediatric T-ALL samples and explore the utility of this functionally relevant molecule as a novel target antigen. Bone marrow (BM) samples of pediatric T-ALL patients (n=54) at time of diagnosis and healthy control individuals (n=14) were collected. We quantified surface expression of CD28 and 25 additional co-stimulatory or co-inhibitory molecules by flow cytometry after gating on T cell precursors (living singlets, CD45 dim/SSC-A low and CD7 +). Subsequently, we generated a set of 20 CD28 directed second-generation chimeric antigen receptors (CARs) based on five different monoclonal antibodies with variation in hinge domain and light chain / heavy chain chronology. We analyzed specific CAR activity against CD28 expressing T-ALL cell lines after retroviral transduction into primary human T cells. Additional CD28 knockout (KO) by CRISPR/Cas9 could prevent T cell fratricide. CAR constructs were subjected to in vivo testing in a T-ALL NSG mouse model with transplantation of 7.5e4 CCRF-CEM cells transduced with firefly luciferase on day 0 and transfer of 2.5e6 CD28 KO T cells on day 3 harboring either of both CD28 CAR molecules. We used CD7 CAR T cells as positive control, CD19 CAR T cells and CD28 KO T cells without CAR as negative controls. We observed significant upregulation of CD28 on T-ALL leukemia when compared to healthy BM donors (Figure 1A, mean 68.8% vs. 3.7%, p=0.0002). We confirmed upregulation of CD28 in published T-ALL RNA-expression data sets. Interestingly, other co-stimulatory molecules such as CD127 were upregulated, too (mean 50.0% vs. 14.6%, p=0.0001), while co-inhibitory molecules such as CD160, TIGIT and TIM-3 were strongly downregulated (>10fold and p<0.0001 each). We hypothesized that this could imply a functional relevance of CD28 in T-ALL. Therefore, we performed co-culture assays of monocyte derived dendritic cells (CDs) expressing CD28 ligands CD80 and CD86 with different T-lineage leukemia cell lines. The presence of DCs significantly increased proliferation of CD28 + T-ALL cell lines under stress conditions. In order to test the feasibility of CD28 CAR T cells, we generated CD28 KO primary T cells without CAR. When tested in co-culture assays with leukemia cells and T cell engagers, CD28 KO T cells showed unchanged cytotoxic capacity and target-dependent activation illustrating that CD28 KO T cells retain short-term effector functions. Based on cytotoxic capacity and CAR T cell expansion, we identified two lead CD28 CAR candidates out of a pool of 20 CD28 CARs. After CD28 KO, CD28 CAR-T cells showed in vitro expansion comparable to CD19 CAR T cells. Next, we asked how CD28 CAR T cells compare to CD7 CAR T cells, which are currently evaluated clinical trials for T-ALL therapy. Therefore, we generated CD7 KO T cells that were subsequently transduced with a functional CD7 CAR. In vitro, both CD28 CAR T cells and CD7 CAR T cells showed killing of CD28 +CD7 + CCRF-CEM cells of >90% at an effector:target (E:T) ratio of 0.2:1, with CD7 being outperformed by one of the CD28 CARs while outperforming the other CD28 CAR at an E:T ratio of 0.04:1 (Figure 1B). Finally, we went on to validate the functionality of CD28 CAR T cells in vivo. We observed significantly prolonged survival of mice treated with CD28 CAR T cells and CD7 CAR T cells when compared to CD19 CAR T cells or control T cells. No difference in survival between CD7 and CD28 CAR T cell treated mice could be observed in two independent experiments. We identify CD28 as novel target antigen for pediatric T-ALL and provide evidence that CD28 is an immunotarget with functional relevance for T-ALL. We demonstrate the feasibility of CD28 CAR T cell generation and that these novel CAR T cells perform equally well as CD7 CAR T cells both in vitro and in vivo. Novel and functionally relevant CAR targets will facilitate clinical development of immunotherapy for T-lineage malignancies.

Transient Differentiation-State Plasticity during Initiation of Pediatric Acute Lymphoblastic Leukemia

Introduction Leukemia is characterized by oncogenic lesions that result in a block of differentiation while at the same time intratumor phenotypic plasticity is retained. It is unclear how these two phenomena arise during leukemogenesis in humans. Here we characterized T-cell acute lymphoblastic leukemia (T-ALL), as a model disease to investigate the coherence between leukemia initiating cells (LICs), developmental arrest, and phenotypic plasticity. Methods We characterized 31 primary T-ALL samples for differentiation state heterogeneity using multi parameter flow cytometry. Subsequently, for 5 patients, phenotypic subpopulations were single cell sorted. As leukemic cells do not expand in vitro primary template-directed amplification (PTA) was used to amplify the genome of single blasts to obtain enough DNA for WGS analysis with nucleotide resolution. Shared and unique mutations were used to construct retrospective lineage trees and assess the hierarchy of phenotypically diverse blasts. Results To assess phenotypic differentiation state heterogeneity in T-ALL, we designed a 17-parameter flow cytometry panel to discriminate T-lymphoid developmental cell populations. We characterized 31 samples (median blast count 98% (IQR = 94 - 99)), and defined immunophenotypic subpopulations as populations with a size of >10% of the total CD7+ population. Differentiation state heterogeneity (> 2 subpopulations) was observed in 26 out of the 31 T-ALL patients (83.9%). Interestingly, the most immature CD4-CD8-CD3- “DN-like” differentiation state was found in all samples, suggesting that developmental hierarchy is maintained. We then questioned whether genetic determinants were driving the phenotypic diversity we observed. Therefore 5 T-ALL patients harboring translocations of oncogenes commonly seen in T-ALL were selected, namely, TLX1 (n=1) , LMO2 (n=2) , TAL1 (n=2) and TLX3 (n=1) . For each patient cells of each differentiation state were single cell and bulk sorted for single cell WGS and bulk WGS, respectively. Structural variant analysis confirmed presence of the oncogenic translocation in all single blasts, validating their (pre-)leukemic origin. Moreover, driver analysis showed that type B mutations in e.g., NOTCH1 and PHF6 were shared among all blasts. Interestingly, there were no potential genetic drivers found that were unique to a subpopulation. Neutral passenger mutations were therefore used to construct phylogenetic trees. Herein, blasts with similar phenotypic differentiation states were genetically closer related than blasts of different phenotypes, revealing the heritability of phenotypes (Fig. 1A). The biased distribution of immunophenotypes across the different branches of the phylogenetic tree was then confirmed in bulk WGS samples of sorted phenotypic populations. Suggesting that switching between phenotypes is not a frequent process. Next, the plasticity of T-ALL was assessed by identifying the phenotypic cell state of the LIC. We assessed V(D)J recombination per single blast and identified monoclonal V(D)J recombination for most of the blasts. Moreover, in TAL1 T-ALL we observed monoclonal TRG, TRB and TRA translocation. This suggested that the LIC had undergone complete V(D)J recombination and was a more mature T-cell progenitor. However, blasts with an immature CD4-CD8- (DN) phenotype had undergone the same V(D)J recombination suggesting phenotypic de-differentiation of the LIC. Additionally, in TLX1 T-ALL, the blasts with the more mature CD4+CD8+ double positive (DP) phenotype had monoclonal TRG and TRB gene recombination, but polyclonal TRA recombination, whereas cells with a more immature DN phenotype had no TRA recombination (Fig. 1B). This indicated an immature T-cell progenitor as the LIC and continued differentiation after leukemia initiation resulting in DP cells with unique TRA recombination. Conclusion In conclusion, we show immunophenotypic differentiation state heterogeneity in diagnostic primary T-ALL samples. By coupling phenotypic data with novel single cell whole genome sequencing techniques, we identified heritability of phenotypic cell states. Moreover, we reveal the ability of the LIC to differentiate and de-differentiate after leukemia initiation. Taken together our results demonstrate a transient period of plasticity during leukemia initiation where phenotypic switches appear unidirectional.

CXCR3/CXCL10 Signaling Pathway Drives T-ALL into the CNS/Meningeal Microenvironment

Cure rates for T-cell acute lymphoblastic leukemia (T-ALL) have improved significantly, but central nervous system (CNS) involvement leads to a poor prognosis. Understanding the mechanisms underlying CNS involvement could lead to more effective therapies. The CXCR3/CXCL10 signaling pathway mediates T cell movement across the blood-CSF barrier during neuroinflammation, but its role in T-ALL colonization of the CNS/meninges remains unclear. In our study, we hypothesized that T-ALL cells exploit normal T-cell function to infiltrate the meninges. We examined CXCR3 expression in thymic CD4+CD8+ double positive (DP) cells of ∆E-NOTCH1 T-ALL and control mice. Levels of CXCR3 were higher in CD4 +CD8 + DP cells of T-ALL mice compared to controls. Furthermore, leukemic cells in the meninges, bone marrow (BM), and thymus had higher CXCR3 levels compared to other organs ( p < 0.01), suggesting an organ-specific CXCR3 expression in infiltrating T-ALL cells. Additionally, primary T-ALL samples (n = 24) had elevated CXCR3 mRNA levels compared to normal thymocytes. Using CRISPR-Cas9, we deleted CXCR3 in the KOPTK1 and PER117 cell lines and in two primary patient samples. CXCR3 deletion resulted in decreased proliferation, delayed cell cycle progression in S/G2/M phases but did not affect apoptotic cell death. CXCR3 inactivation led to decreased activation of AKT, ERK1/2, JNK, and reduced the expression of cell motility regulators. Notably, T-ALL cells migrated more efficiently to CXCL10 than to CXCL9 and CXCL11, which was reduced upon CXCR3 silencing ( p < 0.01). Mechanistically, we demonstrated that USP7, a ubiquitin-specific protease, regulates and stabilizes the CXCR3 protein via deubiquitination. To explore the role of CXCR3 in T-ALL in vivo, we transplanted CXCR3 KO and control KOPTK1 cells into NSG mice (8 mice/group). Mice with CXCR3 KO had increased survival and significantly lower leukemia burden in the BM and extramedullary tissues compared to the control group ( p < 0.01). We next analyzed the levels of CXCR3 ligands, CXCL9, CXCL10, and CXCL11, in the blood serum and CSF of ∆E-NOTCH1 and control mice (5 mice/group). T-ALL mice had elevated CXCL10 levels in both blood serum and CSF, with higher levels in the CSF, suggesting compartmentalization of the immune response between the CSF and blood ( p < 0.01). CXCL9 and CXCL11 expression were minimal or undetectable. Remarkably, upon injecting ∆E-NOTCH1-transformed hematopoietic progenitors into CXCL10 knockout ( CXCL10 KO) and control B6 mice (5 mice/group), tissue analysis revealed a significant decrease in T-ALL meningeal infiltration in the CXCL10 KO mice, but not in other organs ( p < 0.01). Conversely, control mice exhibited high levels of leukemic cells in all tested organs. We found that meningeal stromal cells (PDGFR+, CD13+, CD31-, CD45-) expressed CXCL10 in ∆E-NOTCH1 mice, unlike hematopoietic (CD45+) and endothelial cells (CD31+, CD45-), compared to control mice. We further observed upregulation of CXCL10 in human meningeal stromal cells co-cultured with KOPTK1, PER117, and primary samples. Treatment with a CXCR3 antagonist or knockout of CXCR3 reduced leukemic cell migration to the meningeal stromal cells compared to controls ( p < 0.01). Conversely, migration of T-ALL cells to the tested stromal cells was reduced upon treatment with a CXCL10 neutralizing antibody or by CXCL10 deletion in stromal cells. Additionally, we identified that the CXCR3/CXCL10 signaling pathway regulated the expression of VLA-4 and VCAM-1, promoting T-ALL cell adhesion to the meningeal stroma. Genetic inactivation or pharmacological inhibition of CXCR3 in T-ALL cells or CXCL10 in meningeal stromal cells reduced cell-cell adhesion and the expression of VLA-4 and VCAM-1 in T-ALL and meningeal stroma, respectively, further supporting the role of CXCL10-CXCR3 in regulating T-ALL adhesion. Lastly, we investigated whether inflammatory cytokines derived from T-ALL induce CXCL10 in the meningeal microenvironment. We found elevated expression of Ifng, Tnf and Il27 in T-ALL cells infiltrating the meninges, but not other organs of ΔE-NOTCH1 mice ( p < 0.01). Silencing these genes in human T-ALL cells resulted in reduced CXCL10 expression and secretion, along with reduced leukemic cell migration to the tested meningeal stromal cells. In summary, our study emphasizes the key role of CXCR3-CXCL10 signaling in T-ALL progression and meningeal infiltration.

A Dual Role of SOX11 Expression in the Formation of T-Cell Acute Lymphoblastic Leukemia in Mouse Models

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Intensified chemotherapy and bone marrow transplants have improved the survival of T-ALL patients. However, around 20% of patients still suffer from primary resistant or relapsed T-ALL. Therefore, improving our knowledge of T-ALL biology and developing precision oncology therapeutics is essential for further progress in T-ALL treatment. SRY-related HMG-box 11 (SOX11) is a transcription factor that regulates the development and survival of multiple embryonic tissues and is crucial for the nervous system. SOX11 is expressed in a multitude of tumor types, where it has been associated with either increased or decreased cell proliferation, metastatic potential, and with either good or bad prognosis, depending on cancer type. Despite being absent in the T-cell lineage, SOX11 is expressed in a subset of T-ALLs, where it plays an unknown role. Based on having a malignantly restricted expression, we hypothesize that SOX11 might be oncogenic in T-ALL. Aim: Identify the role of aberrant SOX11 expression in (pre)leukemic T-cells. Methods: Using a conditional Rosa26-SOX11 overexpression mouse model, we generated mice with T-cell specific (using Lck-cre) SOX11 expression (SOX11 Lck). To assess the self-renewal potential of SOX11 Lck thymocytes, we performed thymic transplantations on sublethally irradiated mice expressing a different allele of CD45. To study SOX11 in a leukemic context, we crossed the SOX11 Lck mice with two established T-ALL mouse models that are either driven by the overexpression of Lmo2 (SOX11 Lck-Lmo2 CD2) or by the loss of the tumor suppressor Pten (SOX11/Pten Lck). An aging cohort of these T-ALL models was followed, and tumor samples were collected. Thymi of 8-weeks old mice were used to study the impact of SOX11 in a pre-leukemic context, and T-cell tumors arising in these models were also used for RNA-sequencing. Using a retroviral strategy, we overexpressed SOX11 in human cord blood stem cells, which we differentiated towards the T-cell lineage ex vivo by co-culturing them on OP9 stromal cells expressing Delta-like Notch ligand 4. Furthermore, we transduced Jurkat cells with a SOX11 expression vector, or with an empty vector, and injected the cells into immunocompromised mice to assess the effect of SOX11 on tumor growth in vivo. Results: In SOX11 Lck mice, we found a significant increase in the immature double-negative 3 (DN3) thymic fraction, with a concomitant decrease in the amount of more mature double-positive CD4 +CD8 + (DP) thymocytes (Fig. A). A possible explanation for this phenotype would be the establishment of a stem-like self-renewal program similar to the one driven by Lmo2. However, by performing thymic transplantations into sublethally irradiated mice, we concluded that SOX11 does not confer self-renewal properties to thymocytes. Additionally, we aged a cohort of SOX11 Lck mice and did not observe formation of T-cell malignancies up to 500 days of age. Given that the expression of SOX11 does not suffice to produce T-cell tumors, we generated the SOX11 Lck-Lmo2 CD2 and SOX11/Pten Lck T-ALL mouse models. In SOX11 Lck-Lmo2 CD2 mice, we found a synergistic effect of the co-expression of both genes on the expansion of DN3 thymocytes (Fig. A), which led to accelerated leukemia formation (Fig. B). By sequencing the RNA of pre-leukemic and leukemic DN3 cells, we observed that SOX11 is mostly regulating cell adhesion, morphology, and cell-cell interactions to promote this oncogenic phenotype in Lmo2-driven T-ALL. On the other hand, SOX11/Pten Lck mice suffered from delayed leukemogenesis (Fig. B), and this tumor suppressor role might be associated with the decrease in DP cells observed in the pre-leukemic stage. Lastly, we set out to validate these findings in a human context. The ex vivo differentiation of human hematopoietic stem cells toward the T-cell lineage showed that SOX11 strongly accelerates early commitment to double-negative T-cell precursors. Also, we observed that Jurkat T-ALL cells engraft faster in immunocompromised mice when expressing SOX11. Conclusions: By generating multiple models of SOX11 expression in the T-cell lineage, we showed that SOX11 impacts T-cell differentiation, both in mice and humans, and that it can promote or hinder T-ALL formation depending on the oncogenic driver.

The role of daratumumab in relapsed/refractory CD38 positive acute leukemias—case report on three cases with a literature review

Primary refractory or relapsed T-cell acute lymphoblastic leukemia (T-ALL) and mixed phenotype myeloid/T-cell acute leukemia have dismal prognoses. New treatment approaches, preferably targeting specific leukemic aberrations to overcome resistance, are urgently needed. The bright expression of the CD38 antigen found in several cases of T-ALL led to an investigation into the role of anti-CD38 antibodies in the treatment of T-ALL. Here, we present three cases of resistant and relapsed T-ALL and myeloid/T-cell treated with daratumumab-based therapy, including venetoclax and bortezomib (Dara-Ven-Bor). All patients achieved complete remission, with minimal residual disease negativity within four weeks of treatment, allowing them to proceed to allogeneic hematopoietic cell transplantation. The toxicity of the triple schema was acceptable. Our patients and other cases reviewed here suggest that daratumumab combined with venetoclax and bortezomib may be a very effective and relatively safe salvage treatment, even in primary resistant T-ALL.

Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia

We previously found that T-cell acute lymphoblastic leukemia (T-ALL) requires support from tumor-associated myeloid cells, which activate Insulin Like Growth Factor 1 Receptor (IGF1R) signaling in leukemic blasts. However, IGF1 is not sufficient to sustain T-ALL in vitro, implicating additional myeloid-mediated signals in leukemia progression. Here, we find that T-ALL cells require close contact with myeloid cells to survive. Transcriptional profiling and in vitro assays demonstrate that integrin-mediated cell adhesion activates downstream focal adhesion kinase (FAK)/ proline-rich tyrosine kinase 2 (PYK2), which are required for myeloid-mediated T-ALL support, partly through activation of IGF1R. Blocking integrin ligands or inhibiting FAK/PYK2 signaling diminishes leukemia burden in multiple organs and confers a survival advantage in a mouse model of T-ALL. Inhibiting integrin-mediated adhesion or FAK/PYK2 also reduces survival of primary patient T-ALL cells co-cultured with myeloid cells. Furthermore, elevated integrin pathway gene signatures correlate with higher FAK signaling and myeloid gene signatures and are associated with an inferior prognosis in pediatric T-ALL patients. Together, these findings demonstrate that integrin activation and downstream FAK/PYK2 signaling are important mechanisms underlying myeloid-mediated support of T-ALL progression.

Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells

BackgroundPatients with relapsed/refractory T-cell malignancies have limited treatment options. The use of chimeric antigen receptor (CAR)-T cell therapy for T-cell malignancies is challenging due to possible blast contamination of autologous...

USP1 promotes the aerobic glycolysis and progression of T-cell acute lymphoblastic leukemia via PLK1/LDHA axis.

The effect of aerobic glycolysis remains elusive in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Increasing evidence has revealed that dysregulation of deubiquitination is involved in glycolysis, by targeting glycolytic rate-limiting enzymes. Here, we demonstrated that upregulated deubiquitinase ubiquitin-specific peptidase 1 (USP1) expression correlated with poor prognosis in pediatric primary T-ALL samples. USP1 depletion abolished cellular proliferation and attenuated glycolytic metabolism. Invivo experiments showed that USP1 suppression decreased leukemia progression in nude mice. Inhibition of USP1 caused a decrease in both mRNA and protein levels in lactate dehydrogenase A (LDHA), a critical glycolytic enzyme. Moreover, USP1 interacted with and deubiquitinated polo-like kinase 1 (PLK1), a critical regulator of glycolysis. Overexpression of USP1 with upregulated PLK1 was observed in most samples of patients with T-ALL. In addition, PLK1 inhibition reduced LDHA expression and abrogated the USP1-mediated increase of cell proliferation and lactate level. Ectopic expression of LDHA can rescue the suppressive effect of USP1 silencing on cell growth and lactate production. Pharmacological inhibition of USP1 by ML323 exhibited cell cytotoxicity in human T-ALL cells. Taken together, our results demonstrated that USP1 may be a promising therapeutic target in pediatric T-ALL.

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

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL. The early T-precursor (ETP-ALL) subset is characterized by an immature T-cell phenotype, chemoresistance, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in half of T-ALLs, particularly those with an immature T-cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is essential for ETP-ALL cell survival. Here, we show that MERTK and BCL-2 mRNA and protein are preferentially expressed in ETP-ALL patient samples. The dual MERTK/FLT3 inhibitor MRX-2843 decreased MERTK activation and downstream signaling, inhibited cell expansion, and induced cell death in ETP-ALL cell lines. Further, 54% (21/39) of primary T-ALL patient samples were sensitive to MERTK inhibition. Treatment with MRX-2843 significantly reduced leukemia burden and prolonged survival in cell-line-derived T-ALL and ETP-ALL xenograft models. In a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 markedly reduced peripheral blood leukemia and spleen weight compared to vehicle-treated mice and prolonged survival. MRX-2843 also synergized with venetoclax to provide enhanced anti-leukemia activity in ETP-ALL cell cultures, with a dose ratio of 1:20 MRX-2843:venetoclax providing optimal synergy. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL and provide rationale for clinical development. MRX-2843 monotherapy is currently being tested in patients with relapsed leukemia (NCT04872478). Further, our data indicate that combined MERTK and BCL-2 inhibition may be particularly effective for treatment of ETP-ALL.

DNMT3A Mutations Enhance T-ALL Survival, Which Is Reversed By JAK/STAT Inhibition

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm of T-cell progenitors, which accounts for approximately 15% and 25% of pediatric and adult acute lymphoblastic leukemia (ALL) cases respectively. In T-ALL, accumulation of genomic abnormalities often leads to the aberrant expression of select groups of transcription factors that result in increased proliferation, cell survival and impaired differentiation of T-cell progenitors, which give rise to this disease The Notch signaling pathway is essential for T-lymphopoiesis and T-cell differentiation from thymic precursors. Moreover, gain-of-function mutations in this pathway act as the major genetic lesions that drive T-ALL development (over 60% of adult cases). The de novo DNA methyltransferase enzyme (DNMT3A) is one of the three genes in mammals encoding for enzymes with DNA methyltransferase activity and mutations in this gene are frequently associated with NOTCH1 mutations in adult T-ALL. Interestingly, DNMT3A mutations are rare in pediatric and adolescent T-ALL. Prognosis of pediatric and adolescent T-ALL cases has seen major improvements during recent years with the introduction of high-dose, multi-agent chemotherapy regimens which has not carried over to adult T-ALL patients. These factors suggest the involvement of genetic differences between adult and pediatric T-ALL patients for the differential clinical outcomes Previous work in our lab has shown that mouse T-ALL cells genetically deficient for Dnmt3a are less apoptotic and have increased JAK/STAT signaling. Here, we demonstrate that DNMT3A-mutant human T-ALL patient derived xenografts (PDXs) are resistant to the chemotherapeutic drugs dexamethasone (DEX) and doxorubicin compared to T-ALL patient specimens with wild-type DNMT3A. However, when primary DNMT3A-mutant human T-ALL cells were treated with dexamethasone or doxorubicin in combination with the JAK/STAT inhibitor ruxolitinib (RUX), we observed the survival benefit of DNMT3A-mutant T-ALL cells were mitigated, suggesting enhanced JAK/STAT signaling promotes survival in T-ALL patient cells with DNMT3A mutations. To investigate the genes and pathways that endows DNMT3A-mutant T-ALL cells chemoresistance, gene expression profiling was performed by RNA-seq on DNMT3A-mutant, and wild-type human T-ALL cells treated with DEX+RUX. Gene set enrichment analysis (GSEA) showed that MTORC1, IL2-STAT5, E2F and G2M pathways were significantly downregulated in DEX+RUX treated DNMT3A-mutant PDX samples. E2F and G2M pathways are associated with cell cycle progression and further analysis of these two pathways revealed BIRC5 as a common gene that is downregulated in both these pathways following DEX+RUX treatment. Furthermore, our RNA-seq analysis demonstrated that the expression of BIRC5 in DNMT3A-mutant PDXs is downregulated upon DEX+RUX treatment compared to the DNMT3A-wildtype group. Moreover, baseline BIRC5 expression is higher in DNMT3A-mutant T-ALL PDXs compared to the DNMT3A-wildtype group. Following treatment with the BIRC5 inhibitor YM155, the viability of DNMT3A-mutant T-ALL cells decrease significantly compared to DNMT3A-wildtype PDXs, which were relatively unaffected. In addition, we observed a similar reduction in cell viability in DNMT3A-mutant PDXs following BIRC5 siRNA nucleofection compared to the DNMT3A-wildtype group. Cumulatively, these studies reveal that enhanced JAK/STAT signaling promotes survival and chemoresistance in T-ALL patient cells with DNMT3A mutations and identify the JAK/STAT target gene BIRC5 as a specific genetic dependency of DNMT3A-mutant T-ALL cells. These data provide a critical first step towards a novel target for precision medicine approaches for this patient group.

CXCR4 Blockade Targets Progression of Primary T Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL), a malignant neoplasm of immature T cells, is particularly aggressive leukemia with no targeted therapies. Large-scale sequencing efforts to define the genetic make-up of tumors both at diagnosis and during relapse have provided little actionable information for therapeutic development or predict relapse. T-ALL is reliant on the growth factors and chemokines from the bone marrow (BM) microenvironment for its growth, survival, and BM retention. To identify possible vulnerabilities, we have previously examined the role of CXCL12, a chemokine produced by the BM niche, in maintaining NOTCH1 (Notch1-DE) mutation-driven T-ALL 1. Targeting CXCL12/CXCR4 signaling after leukemic onset reduced the T-ALL burden in murine disease models, suggesting that the CXCL12-CXCR4 could be a viable therapeutic target in human T-ALL.To test the therapeutic role of CXCR4 in human T-ALL, we generated CXCR4 knockout T-ALL cell lines using CRISPR-Cas9 editing and characterized their growth and survival in vitro and in vivo. Although the effect of CXCR4 targeting on leukemic growth and apoptosis in vitro was modest (Figure 1A-B), animals engrafted with CXCR4 knockout T-ALL cell lines survived significantly longer than mice injected with control leukemic cells (Figure 1C). To investigate the clinical efficacy of targeting the CXCR4/CXCL12 axis in primary human T-ALL, we generated T-ALL patient-derived xenografts (PDXs) mouse models from 5 T-ALL and 2 Early T-cell precursor acute ALL (ETP-ALL) primary samples. Remarkably, treating PDX mice with MDX1338, a fully humanized anti-CXCR4 antibody2,3, significantly extended survival from all tested PDX models independently of their mutational landscape (Figure 1D-E). This increased survival was accompanied by mobilization of leukemic cells into peripheral blood, suggesting that CXCR4 targeting leads to leukemic disengagement from the BM niche, affecting their survival. Furthermore, BM imaging revealed disengagement of leukemic cells from the BM niche. Collectively, this study provides a pre-clinical rationale for therapeutic interventions targeting CXCR4 in human T-ALL. Figure 1: CXCR4 antagonism suppresses human T-ALL in vivo. A-B) Growth curve (A) and level of Annexin V (B) of human T-ALL cell lines CRISPR/Cas9-edited with sgCXCR4 or control sgOR2W5. C) Kaplan-Meier survival curves of recipient mice transduced with sgOR2W5 or two independent sgCXCR4 are plotted. D) Schematic of treatment with aCXCR4 Ab (MDX-1338) or isotype control. E-F) Kaplan-Meier survival graph (left) and peripheral blood levels of hCD45+ of NSG mice engrafted with primary adult (E) and pediatric (F) T-ALL samples and treated with aCXCR4 Ab or isotype control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Opposing Effects of KDM6A and JDP2 on Glucocorticoid Sensitivity in T-ALL

Glucocorticoids (GCs) are a cornerstone of front-line T-cell acute lymphoblastic leukemia (T-ALL) treatment. While mutations in NR3C1, which encodes the GC receptor (GR), and other genes involved in GC signaling occur at relapse, other mechanisms of adaptive GC resistance are largely unknown. In an unbiased in vivo screen to discover GC resistance mechanisms, we transplanted and treated 10 primary mouse T-ALLs initiated by retroviral insertional mutagenesis with the GC dexamethasone (DEX) (Wandler et al., 2020). GR protein expression was reduced in ~40% of resistant clones overall, including those with Nr3c1 mutations. The remaining 60% of primary relapsed/resistant leukemias are a valuable resource for discovering additional mechanisms of adaptive GC resistance. Using shotgun cloning, we identified new and unique retroviral integrations near Jdp2 with a corresponding increase in expression in different relapsed clones from independent recipient mice transplanted with T-ALL 8633. Jdp2 integrations were not present in any of the other 9 leukemias. Jdp2 was identified as a T-ALL oncogene in previous insertional mutagenesis screens and shown to induce de novo GC resistance in zebrafish thymocytes (Mansour et al., 2018). T-ALL 8633 was the only leukemia in our panel with a Kdm6a mutation, and we hypothesized that Jdp2 and Kdm6a might interact to modulate GC sensitivity. To explore a possible functional interaction between Jdp2 and Kdm6a in GC resistance, we over-expressed JDP2 using lentiviral transduction and inactivated KDM6A using CRISPR/Cas-9 in CCRF-CEM human T-ALL cells. We refer to these cells as EV (empty vector controls), JDP (JDP2-expressing), EV/KDM (KDM6A mutant) or JDP/KDM (JDP2-expressing; KDM6A mutant). The experiment was reproduced in a second independent CCRF-CEM subclone with similar findings. JDP2 over-expression alone conferred minimal DEX resistance in CCRF-CEM cells (Figure 1). By contrast, KDM6A inactivation unexpectedly enhanced GC sensitivity. Interestingly, JDP2 over-expression reversed this sensitivity in one doubly mutant S1 subclone (JDP/KDM1) and induced overt DEX resistance in another (JDP/KDM2). DEX exposure induced cleaved caspase 3 (CC3) expression in CCRF-CEM subclones that correlated with drug sensitivity, with EV/KDM cells showing the highest levels of CC3 and cells expressing JDP2 alone the lowest. Together, these data suggest a model whereby loss-of-function mutations in KDM6A enhance the sensitivity of T-ALL cells to DEX-induced apoptosis, which then results in selective pressure favoring outgrowth of resistant clones with elevated JDP2 expression. We next investigated basal and DEX-induced NR3C1 and GR expression levels. Upon exposure to DEX, EV/KDM cells up-regulated NR3C1 mRNA and GR protein levels to a greater extent than EV controls, whereas JDP cells were less responsive (Figure 2). Furthermore, "double-mutant” JDP/KDM subclones generally behaved similarly to the JDP2 over-expressing cells, supporting the idea that JDP2 can overcome the DEX sensitization conferred by KDM6A inactivation. To address the potential clinical relevance of these data, we analyzed genomic data from 103 pediatric ALL patients (Li et al., 2020). Of 9 patients with T-ALL and available paired transcriptome data from diagnosis and relapse, two harbored KDM6A mutations. One of these leukemias acquired a somatic NR3C1 mutation at relapse and the other uniquely showed a ~10-fold increase in JDP2 mRNA expression. This relapsed T-ALL also had reduced NR3C1 expression as compared to the paired diagnostic sample. Altogether, these data implicate elevated JDP2 expression as a driver of intrinsic and acquired GC resistance in T-ALL by blocking the expected increase in GR expression in lymphoblasts upon GC treatment. Our findings are consistent with other recent studies showing that genetic and epigenetic mechanisms that converge on GR signaling play a central role in ALL relapse (Li et al., 2021; Autry et al., 2020). We also unexpectedly found that KDM6A inactivation sensitizes T-ALL cells to GC treatment and hypothesize that this results in strong selective pressure for the outgrowth of GC-resistant clones due to JDP2 over-expression and other mechanisms. The FDA-approved BCL-2 inhibitor venetoclax and H3K27me3 inhibition are rational therapeutic approaches for potentially overcoming adaptive GC resistance in relapsed patients with KDM6A-mutant T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Multi-Omics Reveals Immune Microenvironment Alterations in T-Cell Acute Lymphoblastic Leukemia

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that predominantly occurs in children and young adults. Although intensified multi-agent chemotherapy improved overall survival rates, relapsed/refractory T-ALL remains difficult to treat. In addition, these intensive chemotherapies carry a risk for short- and long-term side effects, especially in children. There is an urgent need for novel therapeutic strategies to improve patient outcome. Targeting the tumor microenvironment has been recognized as a promising strategy in many adult tumors with high neoantigen load, but it is thought to be less effective in pediatric and hematologic malignancies as these carry few genetic mutations. However, comprehensive studies of the immune microenvironment are lacking in many pediatric malignancies, including T-ALL, but are urgently required to identify novel therapeutic targets. The aim of this study is to unravel mechanisms of immune evasion in T-ALL and to establish novel immunotherapeutic targets for the treatment of T-ALL. Methods We performed 5’ single cell transcriptome, surface proteome of ~200 proteins and immune repertoire analyses of ~54,000 cells from 10 primary T-ALL pediatric patient samples at initial diagnosis and 4 normal donors using the 10x Genomics platform. Sequencing data was processed using Cell Ranger (reference genome hg38). Quality control was performed using Scrublet (doublet removal), DropletQC (quality control damaged cells) and Seurat (percentage mtDNA < 10% and genes expressed > 200). RNA counts were normalized with sctransform and surface protein antibody counts were centered log-ratio normalized using Seurat. Non-linear dimensional reduction (UMAP) and differential gene/protein expression was done using Seurat and cell type annotation was done using Azimuth in combination with known marker genes. In addition, we analyzed differential cell type abundance (MiloR), the immune repertoire (Immunarch), transcription factor activity (pyScenic) and ligand-receptor activity (NicheNet). Results Our multi-omics analysis revealed changes in the immune cell type composition in T-ALL associated with an immunosuppressive microenvironment. Specifically, we identified a significant enrichment of non-classical monocytes, atypical memory B-cells and double-negative T-cells (CD3+, CD4-, CD8-) in the T-ALL immune microenvironment compared to the normal immune microenvironment. These CD21-, CD11c+ atypical memory B-cells showed high expression of multiple inhibitory receptors (including PD-1, FCRL3/5 and ILT2), suggesting muted BCR-signaling. Transcription factor analysis showed transcriptional activity of known B-cell regulators SOX5, TBX21 and POU2F2, of which SOX5 is known to be highly expressed during late B-cell differentiation stages including atypical memory B-cells. Double-negative (DN) T-cells, which were oligoclonal, were also overrepresented in the T-ALL microenvironment. Correspondingly, immune repertoire analysis of normal T-cells showed decreased TCR diversity (Chao1 index, p = 0.016, Wilcoxon rank sum test) compared to normal donors. Surprisingly, these DN T-cells displayed features of CD8+ T-cell dysfunction, such as high expression of PD-1, TIM3 and LAG3, as well as features of regulatory T-cells, such as high expression of IL10, ICOS and CTLA4. These cells also demonstrated high transcription factor activity of EOMES, NFATC1 and ETV1, which are all associated with a CD8+ T-cell dysfunctional transcriptional network, as well as FOXP3 transcription factor activity, the master transcription factor in regulatory T-cells. Conclusions Multi-omics profiling of the immune microenvironment in T-ALL revealed the presence of several populations of dysfunctional cells associated with an immunosuppressive microenvironment. Studying ligand-receptor interactions analysis between malignant and immune cells will provide the basis for future (pre)clinical studies leading to the development of novel immunotherapeutic strategies for the treatment of T-ALL.

IRX3 Oncogene Activation in T-ALL through Enhancer Hijacking Caused By fto Intron 8 Deletions

IRX3 is a homeobox transcription factor essential for normal embryonic development but with no detectable expression across the normal T-lineage. In contrast, aberrant expression of IRX3 is observed in ~50% of patients with T cell acute lymphoblastic leukemia (T-ALL). Prior studies have confirmed that IRX3 acts as a potent oncogene through its ability to immortalize HSPCs in myeloid culture, and induce lymphoid leukemias in vivo (Somerville et al. 2018). To date, the mechanism of transcriptional activation for IRX3 in T-ALL is unknown. We have previously identified somatically acquired variants of the noncoding genome that robustly activate oncogenes in T-ALL (Mansour et al. 2014; Rahman et al. 2017). Therefore, we investigated whether aberrations of the noncoding genome are a cause of dysregulated IRX3 expression. Two candidate cis-regulatory elements (cCREs) for IRX3 were identified by analyzing enhancer-promoter loops mapped by HiChIP from the IRX3 positive CUTLL1 T-ALL cell line. The first was situated 3' to IRX3 within FTO intron 8, and the second 5' to IRX3 within the CRNDE/IRX5 locus spanning distances of 0.22 Mb and 0.65 Mb respectively. Generation of a rank ordered gene list by comparing IRX3 negative and IRX3 positive primary T-ALLs (n=118) by RNA-Seq, showed that the expression of genes specifically within these loci were positively correlated with IRX3 mRNA levels, with FTO at rank 64, and IRX5 at rank 109 out of 19,464 total genes. This suggested the presence of a long-range regulatory axis in T-ALL encompassing FTO, IRX3, and IRX5. By analyzing publicly available datasets for genetic aberrations within these loci, we identified 12 T-ALL genomes, 11 of primary patient origin and 1 cell line (ALL-SIL) with heterozygous deletions impinging exclusively on the cCRE within FTO intron 8. No mutations were discovered at the CRNDE/IRX5 locus. Matched expression data were available for 5/12 T-ALL cases, with all 5 exhibiting supraphysiological levels of IRX3 mRNA, including 1 case with detectable monoallelic expression, consistent with the hypothesis that these deletions activate gene expression in cis. The aggregated deletions mapped a minimal region of ~155kb within FTO intron 8 that included both a CTCF and MYB binding site. We designed a ddPCR to delineate simultaneous copy number calls at these two sites in a larger cohort of primary leukemias. Heterozygous deletions were present in 6.2% (10/161) of pediatric and 1.5% (2/137) of adult T-ALL patients at diagnosis. Notably, 4/12 cases only had deletion of the CTCF site with the neighboring MYB site unaffected, thereby implicating the CTCF site mechanistically. To assess causality between the deletions and IRX3 dysregulation, we used CRISPR/Cas9 genome-editing to disrupt the CTCF and MYB sites individually or together in the FTO (wt/wt) IRX3 negative PF-382 T-ALL cell line. Deletion of CTCF and MYB sites together, and CTCF site alone, led to a ≥4-fold increase in IRX3 expression, whereas MYB site deleted clones had no detectable impact on IRX3 expression. Furthermore, chromatin looping data from the IRX3 proximal promoter by UMI-4C between T-ALL cell lines with (n=3), and without (n=3) ∆FTO intron 8 identified a significant increase in promoter contacts with the CRNDE/IRX5 locus positioned 5' to IRX3. Remarkably, this locus harbors a super-enhancer determined by rank ordering of all enhancers (rank 113/23,737 total enhancers), and an actively transcribed noncoding RNA (CRNDE) during normal T cell development. Thus, these data reveal that activation of IRX3 in T-ALL is caused by deletion of a CTCF site within FTO intron 8 and consequential long-range enhancer hijacking. We postulate that similar deletions of the noncoding genome especially those that impinge on CTCF sites, may cause dysregulated oncogene expression over vast genomic distances in both hematological and solid malignancies.

Idasanutlin and Navitoclax Induce Synergistic Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

We report here investigation of the combination of idasanutlin and navitoclax in the context of T-cell acute lymphoblastic leukemia (T-ALL), including detailed mechanistic characterization and in vivo patient derived xenograft results. T-ALL is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. An unmet clinical need remains for the development of better therapies for T-ALL. Notch pathway activation mutations are present in the majority of T-ALL and are thought to induce transformation, in part, through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, and critical to our approach, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these genetic data, we hypothesized that treatment with MDM2 inhibitors, by bypassing the loss of ARF, would increase p53 expression and lead to T-ALL cell apoptosis. To test this hypothesis, we initially treated T-ALL patient derived xenograft (PDX) lines in vitro with single-agent idasanutlin (a second generation MDM2 inhibitor) and observed upregulation of pro-apoptotic p53 target genes Bax and Puma1. However, only modest T-ALL cell apoptosis was observed in response to MDM2 inhibition alone. Based on these observations, particularly the induction of pro-apoptotic p53 targets, we hypothesized that inhibition of pro-survival BH3 domain proteins would synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with navitoclax (ABT-263), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient and dose-limiting thrombocytopenia was observed. Combination therapy with idasanutlin could permit on-target inhibition with tolerable dosing. In vitro treatment of a panel of 5 different PDX lines derived from pediatric and adult T-ALL with dual idasanutlin and navitoclax led to robust cell death in all lines. In addition, treatment of a T-ALL PDX line with a matrix of dose combinations showed strong evidence of synergic activity. Synergy was assessed by ZIP (zero interaction potency) score, a statistical method developed by B Yadav et al. (Comput. Struct. Biotechnol. J. 2015) that compares the change in potency of individual dose response curves in the presence of a second agent. Generally, a ZIP score greater than 5 is considered significant; Idasanutlin and navitoclax combination therapy had a global ZIP score=16-18, with the most synergic dose area ZIP score=40-52. Combined treatment resulted in marked apoptosis, with strong induction of pro-apoptotic p53 targets, including PUMA and BAX. Finally, we assessed the in vivo response of four different T-ALL xenografts to dual treatment. In each case, human T-ALL cells were injected into immunodeficient NSG mice and randomized into treatment groups. Monotherapy with idasanutlin or navitoclax resulted in a modest but significant decrease in T-ALL burden in all cases, with the exception of an ETP T-ALL, which was resistant to idasanutlin monotherapy. A marked response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts. Specifically, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change in tumor burden for synergy interaction. Furthermore, overall survival was significantly increased in the combination treatment group. These data suggest that the rational combination of idasanutlin and navitoclax is highly active, with induction of an apoptotic cell death in T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pharmacological Modulation of CD21 Antigen Density Enhances Chimeric Antigen Receptor (CAR-T) Cell Function

Introduction Antigen density has emerged as an important factor regulating CAR-T cell activity with low antigen density being associated with reduced potency in pre-clinical models across several targets. In clinical trials of CD22 CAR-T cells, the low and heterogeneous CD22 expression in B-ALL enabled CD22lo blasts to escape CAR-T cell detection leading to antigen dim relapses (Nat Med. 2018, 24:20-28). We have previously reported on the development of novel CD21 directed CAR-T cells (CAR21) for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) (Blood. 2021, 138:902). Donor derived CAR21 T cells targeting membrane proximal epitopes and utilizing a Fab-CAR architecture were capable of specific cytotoxicity and cytokine secretion against CD21 positive target cells in vitro and were active in vivo. However, in co-culture assays, CAR21 cells secreted less IL-2 against cell lines and primary tumors expressing CD21 at very low antigen density (<1000 copies/cell). We have shown that CD21 is expressed in ~60% of primary T-ALL samples (n=58) although at low antigen density (mean 1262 copies/cell) compared to more established targets such as CD19. We wanted to explore the impact of this low antigen density on CD21 CAR-T function and whether it was possible to upregulate CD21 density by pharmacological means. Results We have previously shown that CD21 is a direct transcriptional target of the NOTCH-MYC pathway and that PI3K/mTOR inhibition of T-ALL results in activation of this pathway and consequent CD21 up-regulation (Leukaemia. 2013, 27:650). We therefore incubated six T-ALL cell lines with four inhibitors of the PI3K/mTOR/AKT pathway (AZD5363, GDC0941, BEZ235 and rapamycin) for 48 and 72hrs and showed surface CD21 up-regulation by flow cytometry of > two-fold at both time points for all inhibitors, with BEZ235 showing the greatest fold increases across all cell lines. Incubation with the gamma-secretase inhibitor L685 resulted in significantly down regulated CD21 expression across all cell lines in keeping with the known NOTCH regulation of CD21 expression. We were next interested in the persistence of this enhanced CD21 expression after drug withdrawal. In two T-ALL cell lines, PF382 and RPMI8402, the impact of BEZ235, rapamycin and L685 was sustained at 48hrs after washing off. PI3K/ mTOR mediated CD21 manipulation did not occur in a CD21 negative T-ALL cell line, normal donor B and T cells or B-ALL patient samples suggesting the effect was specific to T-ALL. When clinically approved PI3K inhibitors (Idelalisib, Duvelisib, Copanlisib, Everolimus) were used, an increase in CD21 expression was again seen across 5 T-ALL cell lines tested. Greatest fold increases (> 2-fold) in CD21 expression were seen with the dual PI3Kα and PI3Kδ inhibitor copanlisib and the mTOR inhibitor rapamycin. No consistent up regulation of CD7 or CCR9 by flow was seen after 48-hour copanlisib incubation suggesting that the effect was specific to CD21. PI3K inhibition was additionally able to mediate 2-fold increases in CD21 expression in a CD21 low patient-derived xenograft T-ALL sample. Further, in a MOLT-4 murine xenograft model of T-ALL, 2 doses of copanlisib were able to produce a 2-fold increase in CD21 expression. These PI3K-mediated increases in CD21 antigen density translated into improved in vitro CAR21 functionality. Across 3 T-ALL cell lines - MOLT-4, CUTLL-1 and RPMI-8402, 48hr pre-incubation with both rapamycin and copanlisib prior to co-culture with CAR21 or CAR19 cells, resulted in improved CD21 specific cytotoxicity, IFN-G and IL-2 release. However, in the same co-culture assay, when copanlisib was added directly to the co-culture rather than via pre-incubation, there was a significant attenuation of IFN-G and IL-2 production by CAR21 suggesting a direct inhibitory effect on CAR-T effector functions in keeping with the known immunosuppressive effects of PI3K inhibitors. Conclusion In summary, we have shown that CD21 expression in T-ALL can be increased through PI3K pathway inhibition and that enhanced CD21 antigen density through pharmacological priming results in improved in vitro CAR21 efficacy. In vivo models analyzing the impact of copanlisib tumor priming on CAR-T efficacy and persistence are ongoing. Target antigen modulation is a promising strategy for enhancing CAR efficacy and reducing risk of antigen negative relapse in low antigen density targets.

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia

AbstractRelapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.