T-cell Acute Lymphoblastic Leukemia Blasts Research Articles

Vinpocetine Mediates Therapeutic Activity Alone and in Combination with Chemotherapy in T-Cell Acute Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive cancer affecting immature T-cells and constitutes about 15% of pediatric acute lymphoblastic leukemia (ALL) cases. Despite advances in treatment, 20% of patients have or develop treatment-resistant disease, and their survival rates are only 15-25% even with additional intensive chemotherapy and hematopoietic stem cell transplant. Furthermore, the current intensive treatment regimens have long-term side effects in most patients. Therefore, there is a pressing need for new, more targeted, and less toxic treatment options for children with T-ALL. Genomic analysis of leukemia samples has enabled identification of chromosomal aberrations and gene mutations associated with T-ALL leukemia development and progression. Single cell analysis of genes expressed in pediatric T-ALL bone marrow samples at diagnosis identified blast-associated signature genes that correlated with poorer event-free survival, suggesting their involvement in leukemia progression and highlighting the potential for further investigation of the signature as an avenue to develop novel T-ALL therapies. A non-conventional genome-oriented approach was applied to the data utilizing the Library of Integrated Network-Based Cellular Signatures (LINCS) resource to identify drugs that are predicted to reverse the T-ALL blast signature. Vinpocetine, a vinca alkaloid derivative, was identified as a potential therapeutic candidate. Methods: Leukemia cell cultures were treated with incremental concentrations of vinpocetine, alone or in combination with a standard chemotherapeutic agent (vincristine or doxorubicin) for 72 hours and relative cell densities were determined by luminescent viability assay. Interactions between drugs were evaluated by mathematical modeling using the fractional product method. Orthotopic T-ALL patient-derived xenografts (PDXs) were established in immune-compromised NSG mice by intravenous injection and peripheral blood samples were collected at intervals for detection of leukemic blasts (human CD45+) by flow cytometry. Mice with low disease burden (<5% peripheral blasts) were randomized to groups and treated every third day with 10 or 20 mg/kg vinpocetine or an equivalent volume of vehicle administered intraperitoneally. Mice were weighed and their health status was evaluated at intervals to monitor for toxicity. Results: Treatment with vinpocetine resulted in a dose-dependent reduction in cell density in T-ALL cell line (Jurkat, HPB, and PEER) cultures relative to vehicle treatment, with IC50 values between 62.6 -64.4 uM, supporting the therapeutic potential of vinpocetine in T-ALL. Vinpocetine also interacted synergistically with vincristine or doxorubicin to provide enhanced therapeutic efficacy against Jurkat cells, indicated by a greater reduction in cell number compared to either single agent. This promising finding underscores the potential of vinpocetine as a supplemental therapy to improve responses or allow for dose reduction of existing chemotherapeutic agents for T-ALL therapy. In a preliminary dose-finding study, treatment with 20 mg/kg vinpocetine was well-tolerated with no overt evidence of toxicity and there was a slight trend toward decreased disease burden. Conclusion: Our study explores the therapeutic potential of vinpocetine for treatment of T-ALL. The observed inhibitory effects on leukemia cell expansion and enhanced efficacy with standard chemotherapies identify vinpocetine as a promising novel treatment approach for T-ALL. Dose escalation studies are needed to evaluate therapeutic effects of vinpocetine in mouse models.

The Atypical Protein Kinase WNK1 Controls Leukemia Progression in TAL/LMO T-Cell Acute Lymphoblastic Leukemia

Genomic and proteomic approaches have been crucial in identifying genetic mutations and protein expression profiles underlying leukemia cell growth and survival, especially in heterogeneous diseases like T-cell acute lymphoblastic leukemia (T-ALL). T-ALL is mainly driven by “poorly druggable” transcription factors along with “fairly druggable” secondary events. However, only a few kinases, such as JAK2 and LCK, have shown dependency for successfully matched therapies, limiting treatment options for T-ALL patients. To identify druggable active pathways in T-ALL, we profiled 31 phosphorylated kinases in T-ALL cell lines, clinical T-ALL blasts, and T cells, and showed that WNK1 exhibited the most significant difference between T-ALL and T cells (p < 0.0001). WNK (with no lysine = K) proteins (WNK1-4) are atypical serine-threonine kinases in which the catalytic lysine, necessary for ATP binding, is replaced by cysteine and situated in subdomain I instead of II. WNK proteins regulate ion flux transport across cellular membranes and facilitate the release/recycling of carrier vesicles; however, their role in leukemia remains uncharacterized. From the analysis of different transcriptome datasets, we demonstrated that WNK1 is the most highly expressed WNK isoform in T-ALL samples, T-ALL cell lines, and patient-derived leukemia xenografts (PDLX) (p < 0.0001). WNK1 and p-WNK1 are predominately expressed in lymphoblasts compared to normal thymus or lymph nodes, indicating the involvement of disease-causing events that lead to the activation of this pathway. To correlate phosphorylation levels with protein and mRNA expression, we intersected the FORALL database, which encompasses ALL's proteomic, transcriptomic, and pharmacoproteomic profiling with the functional Cancer DepMap dataset. WNK1 emerged as a T-ALL lineage transcriptional dependency, with a high correlation between WNK1 protein and mRNA expression (Pearson r= 0.64, p=0.00123, n=22), providing additional support for targeting WNK1 in T-ALL. The next question is whether this level of WNK1 expression can identify a specific T-ALL subgroup. From two independent T-ALL datasets, we found that WNK1 is overexpressed in the TAL/ LMO-related (TAL-R) subgroups (p < 0.01), where PTEN/AKT mutations are notably abundant. An outlier analysis of WNK1 expression revealed a correlation with 6q14-q16 deletions (Fisher's exact test = 0.0268), a privileged abnormality in the TAL/LMO subgroup, suggesting a potential area for clinical exploitation in poor-prognosis T-ALL. To understand the contribution of WNK1 in leukemogenesis, we performed a network-based analysis (NetBID2) from RNA-seq data of T-ALL cells lacking WNK1 expression or treated with WNK463, an orally bioavailable pan-WNKs inhibitor. Results showed that modulation of WNK1 enriched signatures associated with the G2M checkpoint and mitotic spindle control (p < 0.0001). Consistently targeting WNK1 with shRNA or sgRNA-CRISPR Cas9 in TAL-R cell lines promoted polyploidy, resulting in cell cycle arrest and inhibition of cell proliferation in vitro. Similarly, treatment with WNK463 led to morphometric changes, such as incomplete cell division or chromosome segregation through mitotic spindles and abscission defects. Due to the potential translational significance of our findings, we established a PDLX TAL-R model by serially transplanting SIL-TAL1 + leukemia cells into NOD/SCID mice for in vivo drug treatment. Mice (n=10) were exposed to WNK463 at 1.5 mg/Kg/day for 26 days. At the control group endpoint, we sacrificed four mice per group for efficacy studies to reveal a decrease of leukemic cells infiltration in the bone marrow and spleen (p=0.0286), an effect pharmacodynamically associated with a reduction in the WNK1 phosphorylation levels. The remaining mice were observed for survival analysis. Treated mice showed a significant survival advantage (p=0.0009, log-rank Mantel-Cox test) compared to vehicle control. No relevant drug-related toxicity was recorded in the treatment group. In summary, our work demonstrates the significant contribution of WNK1 kinase in the leukemia progression in TAL/ LMO T-ALL cases. The lack of sequence similarity in the ATP binding domain compared to other kinases makes the identification of leukemia's dependence on atypical WNK1 kinase particularly promising for the development of selective inhibitors as a potential treatment for T-ALL.

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...

Pediatric T-cell acute lymphoblastic leukemia blast signature and MRD associated immune environment changes defined by single cell transcriptomics analysis

Different driver mutations and/or chromosomal aberrations and dysregulated signaling interactions between leukemia cells and the immune microenvironment have been implicated in the development of T-cell acute lymphoblastic leukemia (T-ALL). To better understand changes in the bone marrow microenvironment and signaling pathways in pediatric T-ALL, bone marrows collected at diagnosis (Dx) and end of induction therapy (EOI) from 11 patients at a single center were profiled by single cell transcriptomics (10 Dx, 5 paired EOI, 1 relapse). T-ALL blasts were identified by comparison with healthy bone marrow cells. T-ALL blast-associated gene signature included SOX4, STMN1, JUN, HES4, CDK6, ARMH1 among the most significantly overexpressed genes, some of which are associated with poor prognosis in children with T-ALL. Transcriptome profiles of the blast cells exhibited significant inter-patient heterogeneity. Post induction therapy expression profiles of the immune cells revealed significant changes. Residual blast cells in MRD+ EOI samples exhibited significant upregulation (P < 0.01) of PD-1 and RhoGDI signaling pathways. Differences in cellular communication were noted in the presence of residual disease in T cell and hematopoietic stem cell compartments in the bone marrow. Together, these studies generate new insights and expand our understanding of the bone marrow landscape in pediatric T-ALL.

A System-Level Study Reveals That a Defective Thymic Niche Accelerates the T-ALL Initiation

The ontogeny of T-cell acute lymphoblastic leukemia (T-ALL) is unknown. In humans and mice, T-ALL is diagnosed when malignant T cells are detectable in the peripheral blood, i.e., at a stage when disease development is complete. Therefore, the malignant processes in the thymus - where the T cells develop - are an unexplored "black box". We took an interdisciplinary approach combining in vivo experiments with computational modeling to investigate the role of the thymic niche in disease manifestation. We focused on the role of interleukin 7 (IL-7) in the thymus, as approximately 70-80% of T-ALL patients have either increased IL-7 receptor (IL-7R) expression or gain-of-function mutations in this gene. In addition, autocrine production of IL-7 by T-ALL blasts has been detected in some patients. Using our recently developed "virtual thymus" model (Aghaallaei et al., Science Advances, 2021), we first simulated more than 200 scenarios to find out the conditions that lead to massive expansion of a single clone in the thymus. We then genetically engineered thymocytes and thymic epithelial cells (TECs) in medaka fish based on in silico predictions. Consistent with our computational model, we found that manipulation of thymocytes or TECs alone resulted in only 25% (N=36) and 10% (N=29) T-ALL development, respectively, in the thymus of Medaka larvae after five days; however, this frequency was accelerated to 54% (N=45) when both cell types were manipulated simultaneously. Our results strongly suggest that defective thymic niche has a strong impact on T-ALL development. Taken together, we propose a previously unknown mechanism that may trigger T-ALL initiation in the thymus. In addition, we introduce the "virtual thymus" as a new versatile in silico model that can predict the outcome of conditions for the development or treatment of T-ALL.

CD147-specific chimeric antigen receptor T cells effectively inhibit T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is invasive and heterogeneous, and existing therapies are sometimes unsuccessful. Chimeric antigen receptor (CAR) T cell therapy is a breakthrough tumor treatment method, particularly for B cell acute lymphoblastic leukemia. We found that CD147 was highly expressed in tumor T cells of T-ALL patients and T cell lymphoma. Therefore, CD147-CAR T cells that contain a humanized single-chain variable fragment targeting human CD147 and a second-generation CAR frame were constructed for treating T-ALL. CD147-CAR T cells were able to maintain a healthy proliferation rate, preserving a subset of CD62L+/CCR7+ memory T cells. CD147-CAR T cells showed a potent anti-tumor activity against human T-ALL cell line and T-ALL blasts, releasing high level of cytokines in the process. However, CD147-CAR T cells exhibited potential safety toward human normal cells and CD147-deficent cells. NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice were used to establish a T-ALL xenograft model and CD147-CAR T cells conferred robust protection against T-ALL progression and significantly improved survival in mice. Overall, we found that CD147 is a potential antigen target of CAR T cell therapy for T-ALL.

High-valency Anti-CD99 Antibodies Toward the Treatment of T Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain of human CD99, and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.

Imaging flow cytometry reveals a subset of TdT negative T-ALL blasts with very low forward scatter on conventional flow cytometry.

Studies in T-cell acute lymphoblastic leukemia (T-ALL) have shown that leukemic blast populations may display immunophenotypic heterogeneity. In the clinical setting, evaluation of measurable residual disease during treatment and follow-up is highly dependent on knowledge of the diversity of blast subsets. Here, we set out to evaluate whether variation in expression of the blast marker, TdT, in T-ALL blasts could correspond to differences in morphometric features. We investigated diagnostic bone marrow samples from six individual T-ALL patients run in parallel on imaging flow cytometry (IFC) and conventional flow cytometry (CFC). Guided by the imagery available in IFC, we identified distinct TdTneg and TdTpos subpopulations with apparent differences in internal complexity. As TdTneg blasts predominantly displayed very low forward scatter (FSC) on CFC, these subsets were initially excluded from routine analysis as debris, elements of small diameter, apoptotic, and/or dead cells. However, IFC-based morphometric analyses demonstrated that cell size and shape of TdTneg blasts were comparable to the TdTpos cells and without morphometric apoptotic hallmarks, supporting that the TdTneg subpopulation corresponded to T-ALL blasts. Fluorescence in situ hybridization analyses substantiated the clinical relevance of TdTneg FSCvery-low cells by retrieving known diagnostic cytogenetic abnormalities at comparable frequencies in purified TdTneg FSCvery-low and TdTpos FSCint subsets. We highlight this finding as knowledge of phenotypic heterogeneity is of crucial importance in the clinical setting for delineation and quantification of blast subpopulations of potential biological relevance. We argue that the IFC imagery may allow for visual verification and improvement of applied gating strategies.

Characterization of T-ALL-Specific Heterogenous Blast Populations Using High Resolution Single Cell Profiling

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is characterized by proliferation of immature T-cells and accounts for ~15% of pediatric ALL. T-ALL blasts are phenotypically diverse and are sub-classified into pro-, pre-, cortical and mature T-ALL based on the stage of differentiation of the leukemic clone. Early T precursor ALL (ETP-ALL) is a T-ALL subtype associated with higher risk of relapse (Raetz and Teachey, 2016). Bulk sequencing approaches have revealed valuable information about modulated genes in T-ALL; however, little is understood about the interplay between tumor cells and the immune microenvironment. We present a comprehensive single cell RNA sequencing (scRNASeq) analysis of T-ALL samples with the purpose of characterizing the heterogenous tumor and microenvironment cells in order to identify dysregulated genes in leukemia cells and investigate oncogenic signaling pathways. Methods: We profiled 16,280 cells from 5 diagnostic pediatric T-ALL bone marrow samples using the Chromium single cell transcriptomics platform (10x Genomics, Pleasanton, CA). To compare T-ALL versus healthy bone marrow profiles and identify T-ALL-specific malignant blast cell populations, we included data from 3 healthy pediatric bone marrow samples from a recent study (Caron et al, 2020). Dimension reduction using the Uniform Manifold Approximation and Projection (UMAP) approach was used to identify unique cell type clusters (Becht et al, 2018). Using the TARGET dataset (https://ocg.cancer.gov/programs/target), we further evaluated the prognostic significance of identified malignant blast-specific genes by performing Kaplan-Meier survival analysis and compared gene expression patterns and tumor microenvironment makeup between ETP-ALL and non-ETP T-ALL patient samples. Results: We successfully characterized leukemic blasts (CD7+, CD99+ and CD3D+) and other major immune cell types (T cells, B cells, monocytes, erythroid precursors) using the expression of established marker genes (Fig 1A, C). Clustering analysis revealed patient-specific leukemia blast cell clusters (Fig 1B). Differential expression analysis between CD3D+ patient-specific leukemia clusters and CD3D+ clusters comprised of normal T-cells identified a set of 385 promiscuous genes that are significantly differentially expressed between malignant and normal T-cells (p-value &amp;lt;0.05) despite tumor cell heterogeneity. Among the top genes upregulated in the leukemia clusters are HES4 (a downstream target of NOTCH1), CD99, RACK1, and TUBB (Fig 1D), which have previously been implicated in T-ALL leukemogenesis and chemoresistance (DeDecker et al, 2020; Cox et al, 2016; Lei et all 2016). Further, pathways analysis demonstrated significant activation (Z score &amp;gt;1.3, p-value &amp;lt;0.05) of multiple pathways associated with cancer stemness, cellular growth and proliferation including PI3K/AKT, unfolded protein response, and glycolysis, implicating these genes as oncogenic mediators in T-ALL blasts (Fig 2A). Overexpression of S100A4, IFITM2, and CD74 were significantly associated with poor survival in the TARGET cohort of 241 patients (hazard ratio 2.2, 5.8, and 7.1, respectively, p-value &amp;lt;0.05), indicating their prognostic significance (Fig 2B). We additionally evaluated the expression of our gene set across ETP-ALL and non-ETP T-ALL groups in the TARGET dataset. Multiple leukemic blast-specific genes including ARMH1, CD44, CD74, DNAJC1, and IFITM2 were significantly upregulated in ETP-ALL. Further, deconvolution analysis performed on ETP-ALL vs non-ETP T-ALL samples determined that tumor microenvironment cell types are differentially enriched in these T-ALL groups. We observed enrichment of memory B cells, dendritic cells, NK cells, and M2 macrophages in ETP-ALL samples as compared to non-ETP T-ALL samples. Conversely,lower levels of mast cells and T-regulatory cells were observed in ETP-ALL samples. Conclusions: We identified a gene signature characterizing heterogenous T-ALL blast populations. External validation using the TARGET dataset identified genes within the signature that are associated with poor outcomes in T-ALL. Differences in the composition of the immune microenvironment in ETP-ALL versus non-ETP T-ALL samples provide a promising area for future study. Further studies will be carried out with relapsed and non-relapsed T-ALL samples to validate this leukemia signature. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

Multi-Omic Based Antigen Discovery for the Immunotherapy of Pediatric Acute T Cell Lymphoblastic Leukemia

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease due to treatment related complications and poor prognosis of patients with relapsed disease. Immunotherapy with monoclonal antibodies (MAbs) and/or chimeric antigen receptor (CAR) T-cells for T-ALL is limited by identification of tumor specific target antigens. Differential expression is necessary to prevent on-target/off-tumor toxicities and fratricide of activated T-cells. Targeting multiple antigens can bypass immune escape and result in improved T-cell effector function, since antigen density correlates with T-cell activation. Here we designed a pipeline (Figure 1) to identify unique surface antigens expressed in T-ALL using proteomic and transcriptomic analyses followed by flow cytometry validation, and functional studies with CAR T cells targeting the identified antigens. We generated an Illumina total stranded RNAseq library from healthy donor myeloid and lymphoid cells of bone marrow, peripheral blood and cord blood (N= 116). We compared data to 265 St. Jude pediatric T-ALL samples and against 53 normal tissue expression data from the GTEx (Genotype-Tissue Expression) project. To analyze the T-cell surface proteome, we isolated plasma membrane fractions from 11 samples including healthy T-cells and T-ALL cell lines using a differential centrifugation-based method. The purity of the plasma membrane fraction was confirmed by western blot. Na+/K+ ATPase and GAPDH were used as controls for the plasma membrane and cytosolic fractions respectively. Following plasma membrane enrichment, the membrane proteins were applied for proteomic analysis using an advanced TMT-L/LC-MS/MS pipeline, and the acquired proteomic data were further processed via the JUMP software suite. 997 unique proteins were quantified from the membrane fractions. Integrated analysis the transcriptomic and proteomic datasets showed significant correlation and yielded a list of candidate genes, which were validated by flow cytometry on a panel of T-ALL cell lines (CCRF, RPMI8402, and MOLT3) and resting and activated T-cells from healthy donors. We identified GRP78 as one of the differentially expressed cell surface antigens and further confirmed its expression on additional T-ALL cell lines (KE37, PF382, PEER, CEMC7) and 3 PDX samples. Finally, we generated GRP78-CAR T cells and demonstrate that GRP78-CAR T cells recognize and kill GRP78+ T-ALL cells and have potent antitumor activity in xenograft and PDX models. We have established an unbiased pipeline to identify differentially expressed antigens on the cell surface of T-ALL blasts and created a healthy tissue RNAseq library. The results from our analyses are encouraging and interrogation of our pipeline has yielded differentially expressed immunotherapy targets for the treatment of relapsed refractory T-ALL. Our results highlight the importance of integrated surface proteomics and transcriptomics analysis. Figure 1: Outline of strategy for target selection: Figure Disclosures Hebbar: St. Jude: Patents &amp; Royalties. Epperly:St. Jude: Patents &amp; Royalties. Gottschalk:Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; TESSA Therapeutics: Other: research collaboration; Patents and patent applications in the fields of T-cell &amp; Gene therapy for cancer: Patents &amp; Royalties; Merck and ViraCyte: Consultancy. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents &amp; Royalties.

Flow cytometric evaluation of CD38 expression levels in the newly diagnosed T-cell acute lymphoblastic leukemia and the effect of chemotherapy on its expression in measurable residual disease, refractory disease and relapsed disease: an implication for anti-CD38 immunotherapy

BackgroundRecently, anti-CD38 monoclonal antibody (Mab) therapy has become a focus of attention as an additional/alternative option for many hematological neoplasms including T-cell acute lymphoblastic leukemia (T-ALL). It has been shown...

Impact of microRNA-29b on natural killer cells in T-cell acute lymphoblastic leukemia.

Natural killer (NK)-based immunotherapeutic strategies are showing promise in the clinic, particularly against acute myeloid leukemia (AML). Similar treatments for T-cell acute lymphoblastic leukemia (T-ALL) have been less successful, which is due to the higher resistance of T-ALL blasts to the cytotoxic function of NK cells. Herein, microRNA-29b (miR-29b) upregulation was identified in NK cells in both neurogenic locus notch homolog protein 1 (Notch1)-T-ALL mice and patients with T-ALL. Furthermore, miR-29b expression levels were downregulated in T-ALL blast cells. In addition, there was a selective downregulation of an immature subset of NK cells, as well as a reduction in interferon γ (IFNγ) production and natural killer receptor group 2, member D (NKG2D) expression level by NK cells in Notch1-T-ALL mice and patients with T-ALL. Furthermore, when miR-29b knock-out NK cells were adoptively transfused into Notch1-T-ALL mice, partial restoration of IFNγ production and NKG2D expression was observed in NK cells, accompanied by retarded ALL progression and improved survival time. These results implied that T-ALL blast immune evasion occurred via miR-29b-mediated dysregulation in NK cells in the T-ALL microenvironment.

Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia

AbstractRelapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

IL-12, IL-15, and IL-18 pre-activated NK cells target resistant T cell acute lymphoblastic leukemia and delay leukemia development in vivo

ABSTRACTNK cells have shown promise in therapy of hematological cancers, in particular against acute myeloid leukemia. In contrast, the more NK cell-resistant acute lymphoblastic leukemia (ALL) is difficult to treat with NK-cell-based therapies, and we hypothesized that pre-activation of NK cells could overcome this resistance. We show in pediatric and adult patients with T-cell ALL (T-ALL) perturbed NK cell effector functions at diagnosis. Using an in vivo rat model for T-ALL, Roser leukemia (RL), suppressed NK cell effector functions were observed. NK cells from T-ALL patients had reduced expression of the activating receptors NKp46 and DNAM-1, but not NKG2D. In contrast to T-ALL patients, NKG2D but not NKp46 was downregulated on NK cells during rat RL. Decreased frequencies of terminally differentiated NKG2A+CD57−CD56dim NK cells in human T-ALL was paralleled in the rat by reduced frequencies of bone marrow NK cells expressing the maturation marker CD11b, possibly indicating impairment of differentiation during leukemia. RL was highly resistant to autologous NK cells, but this resistance was overcome upon pre-activation of NK cells with IL-12, IL-15, and IL-18, with concomitant upregulation of activation markers and activating receptors. Importantly, adoptive transfers of IL-12, IL-15, and IL-18 pre-activated NK cells significantly slowed progression of RL in vivo. The data thus shows that T-ALL blasts normally resistant to NK cells may be targeted by cytokine pre-activated autologous NK cells, and this approach could have potential implications for immunotherapeutic protocols using NK cells to more efficiently target leukemia.

RSK-mediated nuclear accumulation of the cold-shock Y-box protein-1 controls proliferation of T cells and T-ALL blasts.

Deregulated proliferation is key to tumor progression. Although unrestricted proliferation of solid tumor cells correlates with the cold-shock protein Y-box (YB)-binding protein-1 accumulation in the nuclei, little is known about its expression and function in hematopoietic malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL). Here we show that YB-1 protein is highly enriched in the nuclei of activated T cells and malignant human T-ALL cell lines but not in resting T cells. YB-1 S102 mutations that either mimic (S102D) or prevent phosphorylation (S102N) led to accumulation of YB-1 in the nucleus of T cells or strictly excluded it, respectively. Inactivation of ribosomal S6 kinase (RSK) was sufficient to abrogate T-cell and T-ALL cell proliferation, suggesting that RSK mediates cell-cycle progression, possibly dependent on YB-1-phosphorylation. Indeed, phosphomimetic YB-1S102D enhanced proliferation implying that S102 phosphorylation is a prerequisite for malignant T-cell proliferation. At initial diagnosis of T-ALL, YB-1 localization was significantly altered in the nuclei of tumor blasts derived from bone marrow or peripheral blood. Our data show deregulated YB-1 in the nucleus as a yet unreported characteristic of T-ALL blasts and may refine strategies to restrict progression of hematopoietic tumors.

Pathways for Oncogenesis in T-Cell Acute Lymphoblastic Leukemia Driven By DNA Methylation and Notch Signaling

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia predominantly found in children that has a worse prognosis than other forms of pediatric leukemia with few options for relapsed disease. These issues lead to a need for new targeted therapies. Many of the genes critical for normal T-cell differentiation are aberrantly expressed when patients develop T-ALL, and this knowledge can serve as the basis for rationale design of targeted therapies. In this list are DNA methyltransferase 3A (DNMT3A), a protein that is responsible for de novo DNA methylation, critical for the function of hematopoietic stem cells (HSCs), and NOTCH1, a protein involved in T-cell signaling and regulating cell fate decisions. Patients that with DNMT3A loss-of-functions mutations driving T-ALL have a worse prognosis than their counterparts with normal DNMT3A function. Previous attempts to directly target the action of NOTCH1 have not led to success, specifically gamma secretase inhibitors, so we aim to define the epigenetic regulation of this pathway and the co-operation between DNMT3A and NOTCH1 mutations may open the door for new therapies in patients with the poorest outcomes.We show here that ablation of Dnmt3a in murine HSCs using Mx1-Cre (Mx1-CRE:Dnmt3afl/fl) results in abnormal T-cell development, leading to an accumulation of progenitors at the double negative 2 (DN2) stage (CD4-, CD8-, c-Kit+, CD44+, CD25+) in a proportion of mice (but not all). In affected mice, although there is a significant accumulation of these progenitors, the downstream population of mature T-cells is essentially unchanged compared to wild-type mice. This developmental arrest is associated with increased expression of the Notch1 signaling pathway. Initially suspected as the mechanism given the native action of Dnmt3a, was differential DNA methylation of Notch1 and its downstream targets such as Hes1 and c-Myc, however, through performed bisulfite sequencing of the promoters for those genes and discovered that there was no difference in DNA methylation between wild-type and Dnmt3a-null thymic progenitor cells. The cellular difference underlying accumulation of this Dnmt3a-null thymic progenitor population appears to be increased resistance to apoptosis, and molecular studies are ongoing to explore the molecular mechanism behind this.The observations that thymic progenitor cells lacking Dnmt3a have modified expression of Notch1, and mutations of both DNMT3A and NOTCH1 have been found in T-ALL patient samples, indicates potential co-operation between these two pathways in T-ALL oncogenesis. To test the biological co-operation between these pathways in vivo, we introduced activated Notch1 intracellular domain (NICD) into wild-type and Dnmt3a-null hematopoietic progenitor cells and assessed the latency to T-ALL by bone marrow transplantation. Introducing NICD into a Dnmt3a-null background significantly accelerated T-ALL oncogenesis (median survival wild-type NICD = 72-days, median survival Dnmt3a-null NICD = 46-days, p < 0.001, Figure 1). To explore the molecular differences leading to enhanced T-ALL pathogenesis in a Dnmt3a-null background, we performed RNA-Seq on T-ALL blasts and found that murine leukemia samples with the combination of Dnmt3a loss-of-function and constitutive Notch1 activation show increased in Jak/Stat pathway signaling compared to T-ALL blasts with native Dnmt3a and normal double positive T-cells (CD8+, CD4+).We hypothesize that Jak/Stat pathway inhibitors could improve outcomes for T-ALL patients with combination DNMT3A loss-of-function and activating NOTCH1 mutations. Ruxolitinib is a well-known JAK1/JAK2 inhibitor used in the treatment of myelofibrosis where it has produced robust results. Given the alterations in Jak/Stat signaling, we plan to induce leukemia by elimination of Dnmt3a using the Cre based model, followed by viral introduction of the Notch1 mutant and then treat mice with ruxolitinib and compare survival. It is our hypothesis that the drug will increase latency to leukemia in this quick to develop murine model. Using developed drugs to target new pathways in T-ALL has the potential to offer new strategies to patients with difficult to treat relapsed disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intra-Tumoral Blast Heterogeneity and Implications for Minimal Residual Disease Detection in T-Cell Acute Lymphoblastic Leukemia

Introduction:The early treatment response, measured as minimal residual disease (MRD), is the most important tool for treatment stratification in T-cell acute lymphoblastic leukemia (T-ALL). Flow cytometry-based MRD (Flow-MRD) monitoring, in addition to the PCR-MRD method, is often important to ensure a sensitive MRD marker. Additionally, Flow-MRD investigation may add biological information to the MRD result itself, and allow cell sorting for biological and functional analyses. Flow-MRD in T-ALL consists of identification of cells with immature T-cell phenotype in bone marrow. However, important pitfalls in Flow-MRD, e.g. treatment-related marker modulation and intra-tumoral immunophenotypic heterogeneity, are poorly described. The aim of this study was to explore the implications of these pitfalls on T-ALL MRD detection and on the concordance between the two MRD methods. Potentially both PCR- and Flow-MRD methods might miss blast subpopulations, which is important if subpopulations have divergent chemosensitivity.Methods:The patient cohort included 49 Danish T-ALL patients (1-45 years of age) treated according to the NOPHO ALL2008 protocol. Standard PCR- and flow cytometry-based MRD data were obtained as part of routine MRD monitoring. We investigated intra-tumoral heterogeneity of the leukemia-associated immunophenotype by flow cytometry (diagnostic BM samples), including clonal T-cell receptor gene-rearrangements in flow-sorted blast subpopulations (22 patients). Immunophenotypic MRD markers (including assessment of modulation) were re-evaluated at follow-up in MRD-positive patients. Flow-MRD was validated by PCR-MRD analysis in flow-sorted cell populations (61 follow-up BM samples, 32 patients).Results:At diagnosis, more than 80% of the T-ALL patients had a heterogeneous immunophenotype, most often involving CD1a, CD4, and TdT. The degree of overall heterogeneity, as defined by the number of markers with heterogeneous expression showing distinct blast subpopulations, did not show association to day29 PCR-MRD. Except for one patient, the dominant T-cell receptor clonal gene rearrangements were conserved across phenotypically diverse blasts.Immunophenotypic changes in MRD-positive patients at early follow-up often included subpopulation-loss and/or marker down-modulation of CD1a, TdT and/or CD4. The marker modulations were frequently independent of each other in different subpopulations.Overall, flow cytometry-based identification of blasts and normal cells at Flow-MRD time points was verified by PCR in the flow-sorted cells: In patients where at least 90% of the blasts showed aberrant marker expression at diagnosis, the flow-sorted MRD cells were concordantly PCR-positive, and flow-sorted phenotypically normal cells were similarly PCR-negative in all but three samples that had very high MRD levels (>20%). However, many patients had only partly-informative immunophenotypes (less than 90% of blasts having aberrant marker). Three discrepant cases with Flow-MRD underestimation showed loss of CD1a- and TdT and down-modulation of CD99, verified in flow-sorting experiments.Conclusions and Discussion: We show that intra-tumoral immunophenotypic heterogeneity—a possible result of genetic instability—is common in T-ALL patients and involves several immaturity and T-linage markers commonly used in Flow-MRD. The dominant PCR-MRD targets are in most cases conserved across the diverse blast subpopulations at diagnosis, but in rare cases PCR-MRD might miss a subpopulation. The observed immunophenotypic changes in T-ALL blasts and blast subpopulations at early follow-up, including reduction of immaturity markers, represent important pitfalls in Flow-MRD. Flow-sorting experiments verified that, when all blasts of heterogeneous immunophenotypes were informative, MRD identified by flow cytometry at follow-up was highly concordant with PCR-MRD markers in sorted cells.The T-ALL blast heterogeneity and marker modulations, which are possibly treatment protocol-specific, are important to take into account to obtain reliable Flow-MRD and thus correct treatment stratification of T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Hyperactivation of mTORC1 and mTORC2 by multiple oncogenic events causes addiction to eIF4E-dependent mRNA translation in T-cell leukemia

High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). The activity of the master regulator of this pathway, PTEN, is often impaired in T-ALL. However, experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is required for sustained mTOR activation, even in the absence of PTEN. We previously reported the expression of Neurotrophin receptor tyrosine kinases (TRKs) and their respective ligands in primary human leukemia samples. In the present study we aimed to dissect the downstream signaling cascades of TRK-induced T-ALL in a murine model and show that T-ALLs induced by deregulated receptor tyrosine kinase signaling acquire activating mutations in Notch1 and lose PTEN during clonal evolution. Some clones additionally lost one allele of the homeodomain transcription factor Cux1. All events independently led to a gradual hyperactivation of both mTORC1 and mTORC2 signaling. We dissected the role of the individual mTOR complexes by shRNA knockdown and found that the separate depletion of mTORC1 or mTORC2 reduced the growth of T-ALL blasts, but was not sufficient to induce apoptosis. In contrast, knockdown of the mTOR downstream effector eIF4E caused a striking cytotoxic effect, demonstrating a critical addiction to cap-dependent mRNA-translation. Although high mTORC2-AKT activation is commonly associated with drug-resistance, we demonstrate that T-ALL displaying a strong mTORC2-AKT activation were specifically susceptible to 4EGI-1, an inhibitor of the eIF4E-eIF4G interaction. To decipher the mechanism of 4EGI-1, we performed a genome-wide analysis of mRNAs that are translationally regulated by 4EGI-1 in T-ALL. 4EGI-1 effectively reduced the ribosomal occupancy of mRNAs that were strongly upregulated in T-ALL blasts compared with normal thymocytes including transcripts important for translation, mitochondria and cell cycle progression, such as cyclins and ribosomal proteins. These data suggest that disrupting the eIF4E-eIF4G interaction constitutes a promising therapy strategy in mTOR-deregulated T-cell leukemia.

Targeting components of the alternative NHEJ pathway sensitizes KRAS mutant leukemic cells to chemotherapy

AbstractActivating KRAS mutations are detected in a substantial number of hematologic malignancies. In a murine T-cell acute lymphoblastic leukemia (T-ALL) model, we previously showed that expression of oncogenic Kras induced a premalignant state accompanied with an arrest in T-cell differentiation and acquisition of somatic Notch1 mutations. These findings prompted us to investigate whether the expression of oncogenic KRAS directly affects DNA damage repair. Applying divergent, but complementary, genetic approaches, we demonstrate that the expression of KRAS mutants is associated with increased expression of DNA ligase 3α, poly(ADP-ribose) polymerase 1 (PARP1), and X-ray repair cross-complementing protein 1 (XRCC1), all essential components of the error-prone, alternative nonhomologous end-joining (alt-NHEJ) pathway. Functional studies revealed delayed repair kinetics, increased misrepair of DNA double-strand breaks, and the preferential use of microhomologous DNA sequences for end joining. Similar effects were observed in primary murine T-ALL blasts. We further show that KRAS-mutated cells, but not KRAS wild-type cells, rely on the alt-NHEJ repair pathway on genotoxic stress. RNA interference–mediated knockdown of DNA ligase 3α abolished resistance to apoptotic cell death in KRAS-mutated cells. Our data indicate that targeting components of the alt-NHEJ pathway sensitizes KRAS-mutated leukemic cells to standard chemotherapeutics and represents a promising approach for inducing synthetic lethal vulnerability in cells harboring otherwise nondruggable KRAS mutations.

Class I/II Histone Deacetylase Inhibitors Are a Potential Therapeutic For Notch-1 Driven T Cell Acute Lymphoblastic Leukemia

Abstract Introduction Although T cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease, mutations resulting in activation of the Notch-1 signalling pathway are present in over 50% of patients, thus defining Notch signaling as a central player in T-ALL disease regulation. Furthermore, despite improvement in remission rates following conventional chemotherapeutics, the prognosis for T-ALL remains poor due to disease relapse, which is often refractory to the initial therapies. Inhibitors against the γ-secretase complex, which is part of intracellular Notch-1 (ICN1) signalling, have so far shown limited efficacy and are associated with severe toxicity. Therefore, alternative approaches to treat Notch-1-driven T-ALL are required. The gene expression signature of Notch-1-mutated T-ALLs has revealed downstream activation of numerous pathways, including Myc, PI3K-AKT-mTOR and NFκB, indicating potential sensitivity to small molecule histone deacetylase inhibitors (HDACi). Therefore, the aim of this study was to use a traceable pre-clinical mouse model of Notch-1 driven T-ALL to investigate the potential of HDACi as therapy. Methods Retroviral transduction of mouse hematopoietic stem cells with constructs encoding ICN, followed by transplantation into irradiated recipient mice, is an established model of T-ALL resulting from Notch-1 activating mutations, and closely mimics human disease. For the present study, we transduced foetal liver stem cells from day E14.5 embryos with constructs expressing either ICN1-GFP, or GFP only. Cohorts of recipient mice developed leukaemia as described previously, characterized by splenomegaly, lymphadenopathy, elevated peripheral white blood cell counts, and the presence of double positive CD4+CD8+GFP+ blasts in peripheral blood, with a small fraction of recipients presenting with single positive CD4+GFP+ or CD8+GFP+ blasts. T-ALL blasts were isolated from this primary leukemia colony and transplanted into sub-lethally irradiated recipient mice. Using this model, cohorts of mice rapidly developed onset of leukemia with significant engraftment of T-ALL blasts in bone marrow, spleen, thymus, liver and detectable blasts in peripheral blood 15 days post transplant. Results GFP+ T-ALL blasts were isolated from lymph nodes and spleen and cultured in vitro in the presence of αCD3 and interleukin 2 to induce robust proliferation. Treatment of these T-ALL blasts with the pan-HDACi Panobinostat (LBH-589) induced cell death and inhibited proliferation of remaining viable blasts in a dose-dependent manner. These results illustrate that LBH-589 is a potent inhibitor of survival and proliferation of Notch driven T-ALL at low nM concentrations in vitro. Furthermore, treatment of cohorts of mice transplanted with ICN1-T-ALL with LBH-589 significantly prevented expansion of disease in vivo as determined by white blood cell counts. Additionally, we monitored leukemic cells infiltration in secondary lymphatic tissues using non-invasive GFP imaging. Whilst high GFP readings were observed in vehicle treated groups, we observed very little signal in LBH-589 treated mice confirming prevention of disease expansion. Of most importance, the effect of LBH-589 on progression of disease was reflected in a significant increase in survival of treated cohorts compared to controls. Conclusions The considerably high relapse rates of T-ALL patients demands the development of novel therapies for disease that is refractory to initial therapies. Our results indicate that LBH-589 has strong potential for development of therapies for Notch driven T-ALL and may be a useful addition to current therapies, either during initial treatment or after relapse of chemoresistant T-ALL. Moreover, we have preliminary data suggesting that LBH-589 has the ability to target the predominant oncogenic “Myc signature” associated with constitutive Notch expression. We are currently performing gene and protein expression analysis to exactly determine the molecular effects of HDACi on ICN and associated downstream effector signalling. Disclosures: Johnstone: Novartis: Research Funding.