T-cell Acute Lymphoblastic Leukemia Cell Lines Research Articles

Metabolic Profiling as an Approach to Differentiate T-Cell Acute Lymphoblastic Leukemia Cell Lines Belonging to the Same Genetic Subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor mainly affecting children and adolescents. It is driven by multiple genetic mutations that together define the leukemic phenotype. Interestingly, based on genetic alterations and/or deregulated expression, at least six genetic subgroups have been recognized. The TAL/LMO subgroup is one of the most represented genetic subgroups, characterizing 30-45% of pediatric T-ALL cases. The study of lipid and metabolic profiles is increasingly recognized as a valuable tool for comprehending the development and progression of tumors. In this study, metabolic and lipidomic analysis via LC/MS have been carried out on four T-ALL cell lines belonging to the TAL/LMO subgroup (Jurkat, Molt-4, Molt-16, and CCRF-CEM) to identify new potential metabolic biomarkers and to provide a subclassification of T-ALL cell lines belonging to the same subgroup. A total of 343 metabolites were annotated, including 126 polar metabolites and 217 lipid molecules. The statistical analysis, for both metabolic and lipid profiles, shows significant differences and similarities among the four cell lines. The Molt-4 cell line is the most distant cell line and CCRF-CEM shows a high activity in specific pathways when compared to the other cell lines, while Molt-16 and Jurkat show a similar metabolic profile. Additionally, this study highlighted the pathways that differ in each cell line and the possible enzymes involved using bioinformatic tools, capable of predicting the pathways involved by studying the differences in the metabolic profiles. This experiment offers an approach to differentiate T-ALL cell lines and could open the way to verify and confirm the obtained results directly in patients.

Abstract 4752: Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling

Abstract Introduction Relapsed acute lymphoblastic leukemia (ALL) portends a poor prognosis. Glucocorticoids (GCs) are a central component of a multi-agent regimen to treat ALL. GCs mediate ligand-dependent global transcriptional changes activating feedback loop that increases GC receptor (GR) expression and pro-apoptotic signaling. GC resistance is a common mechanism in relapsed/refractory ALL. Altered function of Enhancer of Zeste Homolog 2 (EZH2), a histone methyl transferase (HMT), including increased expression and activating mutations have been reported in ALL. Previously, EZH2 inhibitors have been shown to increase GC efficacy in non-ALL B-cell lymphoid malignancies including non-Hodgkin lymphoma. Furthermore, EZH2 inhibitors reversed GC resistance and increased efficacy of GCs in an ALL subtype with an activating point mutation in an HMT called nuclear set domain 2. Overcoming GC resistance by novel therapeutic strategies may help improve outcomes in relapsed/refractory ALL. Hypothesis EZH2 inhibitors may enhance GC efficacy by augmenting downstream effects of GCs. Methods We performed in-vitro studies of EZH2 inhibitors, EPZ-6438 and CPI-1205, at graded doses in GC resistance human-derived B-cell (SUPB15) and T-cell (SUPT1, KOPTK1, PEER) ALL cell lines followed by graded doses of dexamethasone (dex). Cell viability was performed using CellTiter-Glo assay. Induction of apoptosis was measured with Annexin V flow cytometry. Pro-apoptotic BIM mRNA and protein levels were measured using real-time PCR and immunoblot. Global H3K27me3 protein levels were assessed using immunoblot. Results GC resistant B-cell ALL and T-cell ALL treated with EZH2 inhibitors for 3-7 days followed by the addition of dex for 48-72 hours led to decreased cell viability and increased apoptosis of the ALL cells compared to cells treated with vehicle (DMSO) and dex alone. EZH2 inhibitors on their own did not cause apoptosis. SynergyFinder revealed Bliss synergy scores between 23- 31 in B-cell ALL and T-cell ALL cell lines with greater than 10 considered a synergistic drug interaction. Immunoblotting of cells treated with EZH2 inhibitors alone showed an increase in pro-apoptotic protein, BIM. BIM level was further enhanced when dex was added in combination with EZH2 inhibitors compared to vehicle and dex alone suggesting that EZH2 inhibitors were augmenting BIM expression. Further assessment of the mechanism of how EZH2 inhibitors lead to improved GC efficacy will be performed by integrating transcriptomic and epigenomic profiling in the presence and absence of EZH2 inhibitors. Furthermore, in-vivo testing of EZH2 inhibitors in patient derived xenografts will be performed. Conclusion EZH2 inhibitors can overcome GC resistance in ALL posing as a potential novel therapeutic agent to treat relapsed/refractory ALL. Citation Format: Mansi Dalal, Joanna Yi, Jianping Li, Amin Shobh, Daphne DupereRicher, Richard Bennett, Jonathan Licht. Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling [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 4752.

Abstract 5920: Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy

Abstract BACKGROUND: Relapse of T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL) post-chemotherapy or post-immunotherapy remains a therapeutic problem warranting novel therapeutic approaches. Aberrant Wnt pathway activation is a common feature in ALL as Wnt activation through β-catenin is involved in self- renewal of drug resistant leukemia cells. A therapeutical approach to target Wnt signaling in T-ALL and B-ALL remains elusive. Here, we focus on the effect of transducin β-like protein 1 (TBL1) inhibitor tegavivint in combination with chemotherapy in T-ALL and B-ALL. We hypothesize that interrupting the binding of TBL1 to β-catenin abrogates leukemia cell survival and test if tegavivint sensitizes these cells to chemotherapeutic agents such as VDL (Vincristine, Dexamethasone, L-asparaginase) as part of preclinical evaluation of tegavivint in T- and B-cell ALL. METHODS: Four T-ALL cell lines one patient-derived T-ALL case, B-ALL cell lines and eight patient-derived B-ALL cases were used in vitro. We performed cytotoxicity studies using Annexin V/7AAD stainings and flow cytometry or Trypanblue counts to exclude dead cells, western blots, and co-immunoprecipitation assays to investigate effects of TBL-1 inhibition using tegavivint on β-catenin-mediated signaling. Tegavivint was injected intraperitoneally along with chemotherapeutic drugs. RESULTS: β-catenin and TBL1 were uniformly expressed in all T-ALL and B-ALL cells as determined by western blotting, albeit at different levels. Tegavivint decreases β-catenin protein expression as seen through western blot as well as decreases the binding of TBL1 to β-catenin as seen through co-immunoprecipitation (co-IP) in both the T-ALL and B-ALL samples. We also determined that tegavivint as a monotherapy induced apoptosis in both cell lines and patient samples at as little of a dose of 5nM after 24 hours of incubation using Annexin-V/DAPI staining by flow cytometry. We observed that tegavivint can sensitize all cases to chemotherapy (V: Vincristine, D: Dexamethasone, L:L-Asparaginase or Peg-Asparaginase) within 24 hours of incubation. Lastly, in vivo evaluation of tegavivint showed that tegavivint alone and in combination with VD chemotherapy significantly extends survival of NSG mice engrafted with patient-derived T-ALL and B-ALL compared to control treated mice. CONCLUSION: Our preliminary data shows that T-ALL and B-ALL are sensitive to TBL1-β-catenin inhibition using tegavivint as a monotherapy. Tegavivint can also sensitize T-ALL and B-ALL to chemotherapy (VDL). As our data supports our hypothesis that TBL-1-β-catenin inhibition is a new target in ALL therapy, further studies are in progress to preclinically evaluate this approach for clinical care. Citation Format: Samantha Hurwitz, Rachel Friedmann, Tatiana Fourfouris, Ki Jun Lee, Zesheng Wan, Chintan Parekh, Fariba Navid, Deepa Bhojwani, Kimberly Holloway, Yong-Mi Kim. Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy [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 5920.

Abstract 5845: Cytarabine resistance leads to a unique mutational signature

Abstract The prognosis of recurrent T-cell acute lymphoblastic leukemia (T-ALL) is poor, mainly due to chemotherapy resistance. The mechanism(s) leading to chemoresistance at relapse are incompletely understood. We previously characterized a mouse strain that is hypomorphic for the DNA replication factor Mcm2. Mcm2hypo mice develop T-ALL due to acquired DNA copy number variations (CNV) involving tumor suppressor or oncogenes. We hypothesize that treatment of Mcm2hypo cells with specific chemotherapy agents may reveal CNV that lead to chemoresistance. We treated two Mcm2hypo T-ALL cell lines (designated 2883 and 2869) and an Mcm2 WT T-ALL cell line (designated 7298) with cytarabine (ARAC), an active agent often incorporated in T-ALL treatment regimens. After one year of treatment with gradually increasing ARAC concentrations, we obtained ARAC resistant cell lines (designated with the suffix CR) that tolerate drug concentrations up to 10,000X that of parental cell lines. These samples were characterized by sparse WGS (for CNV assessment), RNA-Seq and WES. Both the 2883CR and 2869CR cell lines had bi-allelic mutations involving Dck, which is the rate-limiting enzyme in the cytidine salvage pathway, and is thought to be required for metabolism of ARAC. In both cases, one allele was deleted while the second allele had a splice site/region mutation leading to partial intron retention and resultant frameshift. The 7298CR cell line had homozygous deletion of Dck. Western blotting showed absence of WT Dck protein in all three cell lines. Further analysis of WES data revealed thousands of acquired single nucleotide variants (SNV) in both Dck mutant Mcm2hypo cell lines. These mutations occurred in a specific trinucleotide context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, and C>G in a GCC context) generating a mutational signature that is not present in the COSMIC database. In addition, the parental 2883 and 2869 cell lines showed fewer mutations (ie, hundreds vs thousands) in a similar context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, but not the C>G in GCC context). A GEMINI (Genotoxic Mutational Signature Identified After Clonal Expansion In Vitro) assay of untreated 2883CR and 2869CR cells demonstrated that ongoing ARAC exposure was not required for the mutational signature. We hypothesize that the basic signature (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context) can be produced by Mcm2 deficiency, and this signature is amplified and modified (to include a C>G in GCC context) by Dck inactivation. In summary, we identified three mechanisms responsible for Dck inactivation associated with ARAC resistance: copy number loss, splice site mutations, and splice region mutations. Moreover, Dck inactivation leads to a specific mutation signature. These findings add to emerging data that exposure to specific chemotherapy agents can lead to specific mutational signatures. Citation Format: Dengchao Cao, Mianmian Yin, Zhenhua Zhang, Taimour Baslan, Jack Zhu, Ryan Bertoli, Paul Meltzer, Peter Aplan. Cytarabine resistance leads to a unique mutational signature [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 5845.

Abstract 1733: SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia

Abstract The SET-NUP214 (S/N) fusion gene, which results from either cryptic t(9:9) (q34;q34) or del(9) (q34.11q34.13), is found in a very narrow spectrum of patients diagnosed with Acute Myeloid Leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL). AML patients with S/N fusion were reported to have relatively poor prognostic outcomes despite receiving myeloablative conditioning. While previously, the fusion has been described to overrepresent HOX cluster genes, the underlying mechanism of gene regulation that contributes to the pathology remains elusive due to the rare incidence of the disease. The current study used the MEGAL (ACC719, DSMZ) cell line, identified as the solo megakaryocytic AML line expressing the S/N-fusion, for determining genome-wide DNA methylation (DNAm) and gene expression using 935K Infinium MethylationEPIC v2.0 BeadChip array and RNA-sequencing, respectively. The differential DNAm and expression data were analyzed and compared to the hematopoietic stem progenitor cells (HSPC) or normal megakaryocytes (MK). The ChIP-sequencing data from the S/N positive T-ALL cell line LOUCY has been used to represent the coincidence of regulatory chromatin marks and differentially methylated CpG (mC) sites. We observed n=1934 genes upregulated and n=2396 downregulated in MEGAL, compared to both HSPC and MK. The direction of expression changes between MEGAL and HSPC and MEGAL and MK showed a high correlation (r=0.92, p< 0.01). Per the previous literature, we observed an overrepresentation of HOX genes (n=19) in MEGAL. Based on DNAm profiling, we noticed a propensity of hypermethylation in MEGAL across the promoters (93%±0.6), bodies (93%±0.8), and intergenic (85%±0.3) regions, compared to HSPC/MK. Next, we integrated the mC and expression at the promoters and identified that the majority (75%±3) of the genes belong to the hyper-down (n=2563 against HPSC; n=1907 against MK) or hyper-up (n=965 against HPSC; n=923 against MK) clusters. We further investigated the HOXB (HOXB7, HOXB8, HOXB9) and HOXC (HOXC10, HOXC12, HOXC13, HOXC13-AS, HOXC4, HOXC5, HOC6, HOXC8, HOXC9, HOXC-AS1, HOXC-AS2, and HOXC-AS3) cluster genes, where hypermethylated CpGs mainly were concentrated at the CpG islands and overlapped with active promoter (H3K4me3, H3K4me1) signatures or enhancer-like (CTCF bound) elements. We also observed frequent overlaps between mC and H3K36me3 across the observed genes. The differentially expressed genes in this AML subtype were enriched for the hippo-signaling pathway, calcium signaling, focal adhesion, and PI3-Akt pathways. Collectively, our data showed that S/N favors genome-wide hypermethylation, which, in conjunction with the cis-regulatory elements, induces overrepresentation of the HOX cluster genes. These novel epigenetic vulnerabilities are worth pursuing for investigating the cellular function and pathophysiology of the disease. Citation Format: Samrat Roy Choudhury, Akhilesh Kaushal. SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia [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 1733.

In vivo activity of the second-generation proteasome inhibitor ixazomib against pediatric T-cell acute lymphoblastic leukemia xenografts

The overall survival rate of patients with T-cell acute lymphoblastic leukemia (T-ALL) is now 90%, although patients with relapsed T-ALL face poor prognosis. The ubiquitin-proteasome system maintains normal protein homeostasis, and aberrations in this pathway are associated with T-ALL. Here we demonstrate the in vitro and in vivo activity of ixazomib, a second-generation orally available, reversible, and selective proteasome inhibitor against pediatric T-ALL cell lines and patient-derived xenografts (PDXs) grown orthotopically in immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJAusb (NSG) mice. Ixazomib was highly potent in vitro, with half-maximal inhibitory concentration (IC50) values in the low nanomolar range. As a monotherapy, ixazomib significantly extended mouse event-free survival of five out of eight T-ALL PDXs in vivo.

Thymic-Epithelial-Cell-Dependent Microenvironment Influences Proliferation and Apoptosis of Leukemic Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological cancer characterized by the infiltration of immature T-cells in the bone marrow. Aberrant NOTCH signaling in T-ALL is mainly triggered by activating mutations of NOTCH1 and overexpression of NOTCH3, and rarely is it linked to NOTCH3-activating mutations. Besides the known critical role of NOTCH, the nature of intrathymic microenvironment-dependent mechanisms able to render immature thymocytes, presumably pre-leukemic cells, capable of escaping thymus retention and infiltrating the bone marrow is still unclear. An important challenge is understanding how leukemic cells shape their tumor microenvironment to increase their ability to infiltrate and survive within. Our previous data indicated that hyperactive NOTCH3 affects the CXCL12/CXCR4 system and may interfere with T-cell/stroma interactions within the thymus. This study aims to identify the biological effects of the reciprocal interactions between human leukemic cell lines and thymic epithelial cell (TEC)-derived soluble factors in modulating NOTCH signaling and survival programs of T-ALL cells and TECs. The overarching hypothesis is that this crosstalk can influence the progressive stages of T-cell development driving T-cell leukemia. Thus, we investigated the effect of extracellular space conditioned by T-ALL cell lines (Jurkat, TALL1, and Loucy) and TECs and studied their reciprocal regulation of cell cycle and survival. In support, we also detected metabolic changes as potential drivers of leukemic cell survival. Our studies could shed light on T-cell/stroma crosstalk to human leukemic cells and propose our culture system to test pharmacological treatment for T-ALL.

BACH2-mediated CD28 and CD40LG axes contribute to pathogenesis and progression of T-cell lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL characterized by its high heterogeneity and unfavorable clinical features. Despite improved insights in genetic and epigenetic landscapes of T-ALL, the molecular mechanisms that drive malignant T-cell development remain unclear. BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription repressor recognized as a tumor suppressor in B-cell malignancies, but little is known about its function and regulatory network in T-ALL. Here we found extremely low levels of BACH2 in T-ALL clinical samples and cell lines compared to normal T cells. Overexpression of BACH2 in T-ALL cells not only induced cell growth retardation but also inhibited cancer progression and infiltration in xenografts. Further RNA sequencing (RNA-seq) analysis revealed significant alterations in regulation of defense and immune responses in T-ALL cells upon BACH2 overexpression. Strikingly, CD28 and CD40LG, two essential stimulatory molecules on T cells, were for the first time identified as novel downstream targets repressed by BACH2 in T-ALL cells. Interestingly, both CD28 and CD40LG were indispensable for T-ALL survival, since largely or completely silencing CD28 and CD40LG led to rapid cell death, whereas partial knockdown of them resulted in cell-cycle arrest and enhanced apoptosis. More importantly, BACH2-mediated CD28 and CD40LG signals contributed to cell migration and dissemination of T-ALL cells to the bone marrow, thus adding a new layer to the BACH2-mediated tumor immunoregulation in T-cell malignancies.

Suppression of Super-Enhancer-Driven TAL1 Expression By KLF4 in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by differentiation arrest and clonal proliferation of immature thymocytes. Many oncogenes in T-ALL are genes encoding transcription factors. TAL bHLH transcription factor 1 ( TAL1) is one of the most frequently dysregulated transcription factors in T-ALL and is ectopically overexpressed in approximately 50% of T-ALL cases, owing to chromosome translocations, SIL (SCL-interrupting locus) -TAL1 fusion, and 5‘ super-enhancer (SE)-generating mutations. TAL1 overexpression caused by 5‘ TAL1 SE is found in 5% of patients with T-ALL and is associated with unfavorable clinical outcomes. However, no clinically available drugs specifically inhibit TAL1 or TAL1 SE components. Kruppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors that exhibits both oncogenic and tumor-suppressive functions in a context-dependent manner. In T-cell malignancies, KLF4 is regarded as a tumor suppressor because KLF4 expression is silenced by promoter methylation, and the induction of KLF4 promotes apoptosis through the BCL-2/BCL-XL pathway. However, whether KLF4 affects the expression of T-ALL-related transcription factors remains unclear. We first analyzed three independent previously reported microarray datasets to explore the essential downstream targets of KLF4 in T-ALL cells. The following groups of genes were extracted: genes upregulated in patients with T-ALL compared to normal thymocytes (GSE46170), genes upregulated in Klf4-deficient T-ALL mice (GSE75663), and genes downregulated in KLF4-overexpressing human T-ALL cells (Li W et al. Mol. Cancer. 2015). Overlapping genes in these groups were identified as potential downstream targets of KLF4. Among these, we focused on TAL1, a well-known oncogenic transcription factor gene in T-ALL. We performed a functional analysis of KLF4 using TAL1-positive T-ALL cell lines (Jurkat, MOLT-3, CCRF-CEM, and RPMI-8402). Genetic induction of KLF4 showed stronger anti-leukemic effects in Jurkat and MOLT-3 cells, 5‘ TAL1 SE-positive T-ALL cell lines, compared with CCRF-CEM and RPMI-8402 cells, SIL-TAL1-positive T-ALL cell lines. KLF4 overexpression significantly reduced mRNA expression levels of TAL1 in Jurkat and MOLT-3 cells but not in CCRF-CEM and RPMI-8402 cells. Thus, we hypothesized that KLF4 specifically suppressed SE-driven TAL1 expression by impairing mutated TAL1 enhancer activity. The luciferase reporter assay confirmed that the activity of the mutated TAL1 enhancer was significantly reduced by the exogenous expression of KLF4, whereas that of the wild-type reporter was not affected. According to previous studies, mutations in TAL1 enhancers in Jurkat and MOLT-3 cells create MYB primary motifs, and MYB binding at this location is essential for the formation of 5‘ TAL1 SE. Therefore, we examined whether KLF4 represses MYB expression. The chromatin immunoprecipitation assay and qPCR analysis revealed that KLF4 bound to the proximal gene promoter of MYB and directly downregulated its expression. The luciferase reporter assay using the MYB promoter showed a significant reduction of reporter activity upon KLF4 overexpression. Finally, we investigated whether pharmacological induction of KLF4 using APTO-253, a small-molecule inducer ofKLF4, could control leukemia. APTO-253 inhibited the growth of all TAL1-positive T-ALL cell lines at nanomolar concentrations. The GI 50 values of APTO-253 for Jurkat and MOLT-3 cells were lower than those for CCRF-CEM and RPMI-8402 cells, which was consistent with the KLF4-overexpression-induced growth inhibition of T-ALL cell lines. APTO-253 induced KLF4 expression and decreased TAL1 and MYB expression in Jurkat and MOLT-3 cells. Furthermore, exogenous MYB expression rescued growth inhibition by APTO-253. Collectively, these results indicated that APTO-253 suppressed the growth of T-ALL cells by targeting SE-driven TAL1 expression via the induction of KLF4. In summary, we revealed that KLF4 suppressed SE-driven TAL1 expression via the direct inhibition of MYB. Moreover, pharmacological induction of KLF4 demonstrated anti-leukemic activity in T-ALL cells. These findings propose a promising strategy for patients with T-ALL and 5' TAL1 SE.

Combinatorial Optimization of Chemotherapeutic Agents on T-Cell Acute Lymphoblastic Leukemia

Introduction: Up to now, treatment options for T-cell acute lymphoblastic leukemia (T-ALL) remain quite limited. This study aims to assess the efficacy of monotherapies and combination of chemotherapeutic agents for T-ALL. Jurkat, CCRF-CEM are late T-cell progenitor (LTP) cell lines, while Loucy is early T-cell progenitor (ETP) cell line characterized by an immature phenotype. The drugs under evaluation include venetoclax, a selective BCL-2 inhibitor; cytarabine, a cytosine analog with established antineoplastic activity; bendamustine, an alkylating agent; and azacitidine, a DNA methyltransferase inhibitor. Given the notable tumor heterogeneity observed in T-ALL, the comprehensive assessment of these therapeutic agents on Jurkat, CCRF-CEM, and Loucy may render valuable insights for treatment strategies in T-ALL. Methods: Cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. Venetoclax, bendamustine, and azacitidine were prepared as stock solutions in DMSO, while cytarabine was prepared in distilled water. Monotherapies (venetoclax, cytarabine, bendamustine and azacitidine) and combination therapies (venetoclax plus cytarabine, venetoclax plus bendamustine and venetoclax plus azacitidine) were administered. A control group received DMSO or distilled water only. Cell viability was assessed after 48 hours by using Trypan blue and colorimetric method (MTS assay) on cells treated with various drug concentrations. Statistical analysis was conducted using the SynergyFinder Plus and drc R package, with significance set at p < 0.05. Results: Our study demonstrated that T-ALL cell lines displayed a different pattern of drug sensitivities according to combination of chemotherapeutic agents. Loucy showed greater sensitivity to venetoclax while Jurkat and CCRF-CEM exhibited higher sensitivity to cytarabine, bendamustine, and azacitidine. When conducting drug combinations on Jurkat and CCRF-CEM, the addition of venetoclax to cytarabine, bendamustine, or azacitidine resulted in an additive effect, with Loewe synergic scores ranging from -10 to 10 for each combination. On the other hand, Loucy demonstrated a distinct pattern of response; the combination of venetoclax with cytarabine showed additivity (Loewe synergic score: 8.45 and 5.82 with MTS and trypan blue assays, respectively), while venetoclax combined with bendamustine or azacitidine showed a synergistic effect (Loewe synergic score > 10 with MTS assay). Notably, among these combination treatments on Loucy, the pairing of venetoclax with bendamustine showed the most significant synergistic effects, with a Loewe score of 16.6. Conclusion: Venetoclax-based chemotherapeutic combinations exhibited various responses in T-ALL cell lines. Of note, the combination of venetoclax plus bendamustine showed potent synergistic effects in all T-ALL cell lines. These findings warrant further study to develop fundamentally curative therapeutic strategies in T-ALL.

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.

Development of a Novel L-Asparaginase with Enhanced Properties Using Ancestral Sequence Reconstruction

The overall survival of children with acute lymphoblastic leukemia (ALL) now exceeds 90% due to the precise disease risk stratification and intensification of multi-agent chemotherapy regimens. L-asparaginase (L-ASNase) is an indispensable biotherapeutic enzyme used in treatment of pediatric ALL. Discontinuation of L-ASNase treatment due to immunological or adverse reactions leads to significantly inferior disease-free survival (73% ±7% vs 92% ± 2%; P <0.01, Gupta et al. JCO 2020). Clinical L-ASNases are bacterial in origin, derived from either Escherichia coli or Erwinia chrysanthemi and are therefore highly immunogenic. Additionally, emerging data suggests that the concurrent glutaminase activity seen in bacterial asparaginases exacerbates its liver and pancreatic toxicity, especially in adults thereby limiting its extensive use beyond children. Therefore, finding alternative sources with preferable biochemical features remains paramount. The inferior catalytic properties of human L-ASNase restricts its functionality in ALL treatment. However, characterization has shown that guinea pig (GP) L-ASNase demonstrates significant anti-tumor properties and improved enzyme kinetics despite a 69.8% amino acid sequence identity with human L-ASNase, as compared to 30% for bacterial L-ASNases. Additionally, guinea pig L-ASNase has no glutaminase activity. Thus, to overcome the major deficiencies of bacterial L-ASNase and develop a more humanized form of L-ASNase we performed ancestral sequence reconstruction (ASR). ASR is a protein drug discovery / optimization platform that predicts ancient DNA and protein sequences from extant sequences. This approach permits higher-resolution mapping potentially highlighting functional resides. The objective therefore is to exploit functional diversity refined by natural selection using ASR to identify therapeutic variants with superior properties. Recent work on guinea pig (GP) L-ASNase (Schalk et al. J Biol Chem 2014) identified a low micromolar (μM) Michaelis constant (Km) at 57.7 ± 6.4 μM essential for hydrolyzing the extra-cellular pool of asparagine ~50 μM in human blood; thus, equipping us with the ideal mammalian L-ASNase ortholog to serve as a template to resurrect ancient ASNase sequences. Ancestral L-ASNase sequences were generated utilizing 54 extant L- asparaginase sequences, aligned using MUSCLE and an evolutionary tree inferred using MrBayes. A total of 53 ancestral nodes were identified, and the ten ancestral L-ASNase variants spanning the ancient primate and guinea pig (GP) lineage were resurrected. The sequence identity of these ancestral L-ASNase variants ranged from 81 to 98% when compared to human L-ASNase. E. coli codon optimized complementary DNA (cDNA) sequences were synthesized and subcloned intoan expression vector. Correctly cloned plasmids were transformed into E. coli BL21 (DE3) cells for protein expression. An-ASNase candidates were isolated by Ni 2+ affinity chromatography and then further purified by size exclusion chromatography. Asparaginase activity of the An-ASNase candidates were determined via a modified Nessler's reagent assay by using a continuous spectroscopic enzyme-coupled assay. Three of the ancestral L-ASNase candidates, An-88, An-104 and An-107 demonstrated excellent asparaginase activity when tested at an enzyme concentration of 0.1 mg/mL and an excess substrate concentration, with activity within a comparable range to the clinically relevant E. coli and Erwinia asparaginases. An-88 had 81% similarity whereas both An-104 and An-107 ASNases had 88% identity to human L-ASNase, which is in stark contrast to the only 30% similarity seen with bacterial L-ASNases. Detailed enzymatic characterization of these three lead drug candidates is currently being performed. Preliminary cytotoxicity data testing the ancestral L-ASNase candidates against a T-cell ALL cell line CCRF-CEM showed an expected dose response, with An-107 showing the highest cytotoxicity. Our findings demonstrate that ASR is an efficient protein drug design/optimization platform that we have now extended beyond our previous work with recombinant coagulation factors (Zakas et al. Nat Biotech 2017, Knight et al. Blood Adv 2021) to therapeutic enzymes; thereby, enabling the creation of novel recombinant L-ASNases with translational implications due to improved enzymatic specificity and reduced toxicity.

Ubiquitin-Mediated Degradation of MORF4L1 By FBXO11 Suppresses Cholesterol Biosynthesis in T Cell Acute Lymphoblastic Leukemia

The substrate receptor of SKP1-Cullin1-FBOX complex, FBXO11, is frequently mutated in both diffuse large B cell lymphoma and acute myeloid leukemia, and typically functions as a tumor suppressor by targeting substrates for ubiquitin-mediated degradation. Although FBXO11 is also mutated in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines, the underlying mechanism in T-ALL development remains to be established. Here, we identified that both endogenous activation and exogenous overexpression of FBXO11 could suppress the growth of T-ALL cell in vitro and in vivo. While conversely, loss of FBXO11 accelerates the T-ALL cell proliferation. Transcriptomic and total quantitative proteomic analyses in FBXO11-depleted T-ALL cells reveal activation of the cholesterol biosynthesis pathway with accumulation of intracellular cholesterol levels. Importantly, we identified a chromatin regulator MORF4L1, mortality factor 4 like protein 1, as a functional substrate of FBXO11 in T-ALL cells. We first observed that FBXO11 exhibited frequent copy number alterations in T-ALL cell lines and in patient cohorts from public datasets. Moreover, by CRISPR SAM (Synergistic Activation Mediator) endogenous activation and lentiviral mediated gene overexpression, we validated that FBXO11 played a role in inhibiting tumor cell proliferation in T-ALL cells both in vitro and in vivo. Conversely, loss of FBXO11 accelerated T-ALL cell proliferation. Taken together, our results suggest that FBXO11 functions as a tumor suppressor in T-ALL. Mechanistically, we uncovered that the absence of FBXO11 could activate the cholesterol synthesis signaling pathway from both the RNA-seq and the TMT proteomic analysis in FBXO11-depleted MOLT4 cells compared to control cells. Concordantly, loss of FBO11 resulted in the accumulation of intracellular cholesterol level, suggesting that FBXO11 may suppress cholesterol biosynthesis in T-ALL cells. To understand how FBXO11 controls cholesterol biosynthesis, we further identified the potential substrates from the combined quantitative transcriptome and proteome analysis. MORF4L1, a chromatin modifier involved in histone acetylation, was identified as a key substrate of FBXO11 based on its upregulation at the protein level in FBXO11-depleted cells. Moreover, our data suggest MORF4L1 is a regulator of cholesterol biosynthesis in T-ALL. The inhibition of MORF4L1 suppressed the cellular growth of T-ALL cell lines, and led to the downregulation of the expression of cholesterol-related genes, such as gene HMGCR and HMGCS1. Of note, suppression of MORF4L1 and its cholesterol biosynthesis target genes such as HMGCR, HMGCS1 could rescue the accelerated growth effect after loss of FBXO11, indicating a functional role for MORF4L1 in T-ALL development. In summary, our data support a model wherein the degradation of MORF4L1 by FBXO11 suppresses leukemia cell growth by controlling cholesterol biosynthesis. The identification of this regulatory mechanism is required for T-ALL and provides valuable insights into the metabolic vulnerabilities of leukemia.

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 CNS Microenvironment Promotes Leukemia Cell Survival By Disrupting Tumour Suppression and Cell Cycle Regulation in Pediatric T-Cell Acute Lymphoblastic Leukemia

Although modern treatment has improved the overall survival of pediatric T-cell acute lymphoblastic leukemia (T-ALL) greatly, more than 20% of all children diagnosed with T-ALL relapse (Fattizzo et al., 2020). Approximately 30% of all relapse cases occur in the central nervous system (CNS) and CNS involvement is considered a major risk factor for treatment failure and increased mortality (Frishman-Levy & Izraeli, 2017). To date, CNS directed therapy is considered essential for complete cure to prevent relapse in the CNS, leading to severe neurotoxic effects and long-term adverse complications (Thastrup et al., 2022). Due to lack of knowledge regarding the mechanisms promoting leukemia survival in the CNS and absence of reliable biomarkers to predict CNS involvement, the development of new therapeutic drugs able to eradicate leukemia cells in the CNS is not progressing, therefor limiting the overall survival of pediatric T-ALL patients (Thastrup et al., 2022). To identify genetic alterations in CNS involved T-ALL we performed an RNA-sequencing analysis coupled with a gene set enrichment analysis (GSEA) studying pediatric T-ALL patient samples obtained from the TARGET initiative. We included 30 samples from patients at initial diagnosis subdivided by degree of CNS involvement, ranging from CNS1 (no CNS disease) to CNS3 (advanced CNS disease), as well as 13 samples from patients who had relapsed in the CNS. Our analysis identified distinct genetic alterations in CNS involved T-ALL and CNS relapse compared to diagnosis samples without CNS implications, with dysregulation of genes involved in tumour suppression, apoptosis and self-renewal. CNS relapsed patients were distinguished from diagnosis patients by a distinct gene expression pattern, with an upregulation of many genes, including cell cycle inhibitor CDKN2A and transcription factor HOXA3 (Figure 1A). We also found disruption of many signaling pathways, including enrichment of the p53 pathway in patients who relapsed in the CNS compared to non-CNS involved diagnosis patients. To further study the effects of CNS implications in pediatric T-ALL, we established a co-culture of pediatric T-ALL cell lines (n=4) and a glioblastoma cell line (U87-MG). Following co-culture, changes to mRNA expression was evaluated by RT-qPCR, revealing dysregulation of many genes, including an upregulation of cancer stem cell marker and cell adhesion molecule CD44 (Chen et al., 2018). Moreover, T-ALL cells grown in CNS co-culture displayed a downregulation of tumour suppressor TP53 and an upregulation of its negative regulator, proto-oncogene MDM2 when compared to cells grown in suspension (Figure 1B) (Hernandez Borrero & El-Deiry, 2021). Co-culture also induced pro-survival isoform switching of BCL2L1 (BCLX), part of the BCL family of apoptotic regulators (Daniel et al., 2013). Together, these results suggest an increased survival and loss of tumour suppression of T-ALL cells when grown in direct contact with CNS derived cells. To determine effects of the CNS microenvironment on therapy resistance, T-ALL cell lines grown in co-culture were exposed to 2µM methotrexate, a chemotherapeutic agent used during CNS directed therapy (Thastrup et al., 2022). Leukemia cells in co-culture displayed an increased survival compared to cells grown in suspension following 48 hours of methotrexate exposure, as well as dysregulation of several genes involved in cell survival, self-renewal and cell cycle regulation. Cyclins and CDKs involved in controlling cell cycle progression were differentially expressed in co-culture versus suspension, with a downregulation of CDK2 and cyclin A, crucial for S/G2 transition, as well as CDK1 and cyclin B, responsible for G2/M transition (Leal-Esteban & Fajas, 2020). These results indicate a shift towards cell cycle arrest and senescence of T-ALL cells during methotrexate exposure in CNS co-culture, potentially promoting leukemia survival and chemoresistance. Taken together, our findings indicate a differential gene expression pattern in CNS involved T-ALL, which could potentially be utilized as predicative biomarkers to properly identify patients at risk of CNS relapse. CNS co-culture revealed cell cycle alterations and loss of proliferation markers, promoting senescence and therapy resistance, which could serve as potential therapeutic targets.

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced > 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia

T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies. The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity. The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects. First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 >1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 <1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC 50 <1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=0.89, p=0.03), providing a marker of response. We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition. Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores >10), including leukemias being insensitive to single compounds. Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p<0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p<0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

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.

Clonal Evolution Dissection Reveals High MSI2 Level Promotes Chemo-Resistance in T-Cell Acute Lymphoblastic Leukemia

Occurrences of T-cell acute lymphoblastic leukemia (T-ALL) arise in approximately 10-15% of pediatric ALL cases and the outcomes for high-risk T-ALL are inferior. Currently, the precise dissection of clonal evolution with intra-tumoral heterogeneity and to delineate targetable molecular events driving relapse in T-ALL subtypes have become area of interest. We carried out high-throughput droplet-based 5′-single-cell RNA-sequencing (scRNA-seq) and paired T-cell receptor sequencing (scTCR-seq) of isolated CD7 + BMMCs from five patients' Dx_Rel paired samples. To investigate the gene signature variation underlying leukemic progression, we observed two distinct patterns of evolutionary trajectories in the four Dx_Rel nonETP-ALL pairs. From T956 and T723, a significant outgrowth in TCR initially negligible diagnostic subclusters at relapse, to which surrogate TCR repertoires were correspondingly linked and enumerated, suggestive robust dominant “clonal shifts” under continuous chemotherapeutic pressure. Whereas in Dx_Rel pairs T593 and T856, which contained subclusters with distinct gene signatures at diagnosis or relapse respectively, shared an identical TCR genotype (TCR Dx-Rel major clone). The clonal architecture clues gained from simultaneous scRNA-seq and scTCR-seq are strongly indicative of the leukemic “clonal drift” that commonly occurs under chemotherapeutic stress, during which somehow, hypothetically, molecular signatures within the same TCR subclones intrinsically progressed over time ( Fig. A-H). Next, we designed a targeted scDNA-seq library within a custom panel covering 127 amplicons and 102 genes via Tapastri platform to validate these two patterns of clonal evolution during leukemia progression unambiguously. Likewise, we captured that in persisted subclones in T593 and T856 leukemic cells attained reproductive fitness from maintaining a mutational static status with remarkable transcriptomic variation from diagnosis to relapse. To further interpret the transcriptomic signature drift in the persisted TCR-defined major clones from T593 and T856 pairs, differential expression analysis was employed with RNA-binding protein Musashi-2 (MSI2) showing the most prominent differential expression pattern across drifted subclones catching our attention as a potential indicator of intrinsic clonal progression related to chemo-resistance. Comprehensive analysis of RNA-seq data from the CCCG-ALL-2015 cohort (n=72) and TARGET T-ALL cohort (n = 264) revealed that higher levels of MSI2 mRNA expression in pediatric T-ALL were correlated with higher measurable residual disease (MRD) after induction, and significant correlation between elevated MIS2 mRNA and resistance to prednisone, daunorubicin, and cytarabine were observed from ex vivo drug response assay. RNA immunoprecipitation and high-throughput mRNA sequencing from T-ALL cell lines detected a consistent significant enrichment of MYC transcript peaks in three T-ALL cell lines, indicating the molecular mechanism of MSI2 upregulation on MYC in the progression of T-ALL. Finally, In vitro and in vivo pharmacotyping analysis further validated the MSI2 inhibition significantly sensitized T-ALL to the chemotherapeutic drugs. Collectively, our study leveraging single-cell multi-omics profiling has provided a snapshot of the clonal architecture evolution that occurs from diagnosis to relapse in T-ALL, proving the coexistence of “clonal shift” and “clonal drift” in leukemic progression. Systemic analysis of drifted T-ALL clones has provided compelling evidence of the posttranscriptional regulatory effect of MSI2 on MYC and its potential correlation with leukemic reoccurrence, pinpointing MSI2 as a promising drug target of T-ALL in the future practice.