T-cell Acute Lymphoblastic Leukemia Research Articles

NGS-Based Stratification Refines the Risk Stratification in T-ALL and Identifies a Very High-Risk Subgroup of Patients

Purpose We previously reported a significantly better outcome in adult and pediatric T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 ( N/F) mutations without alterations of K-N-RAS and PTEN(R/P) genes . High-throughput next-generation sequencing strategies (NGS) allowed us to refine the prediction of outcome in T-ALL. Patients and Methods 198 adult T-ALLs in first remission (CR1) from the GRAALL-2003/2005 protocol were included in the study as the construction cohort, and 242 pediatric T-ALLs from FRALLE2000 were used as a validation cohort. Targeted whole-exome sequencing of 63 T-ALL-related oncogenes was performed. Primary outcome was cumulative incidence of relapse (CIR). To account for the large number of candidates genes, selection was performed using a LASSO penalization in a Fine and Gray model predicting CIR (Fu Z. et al. Lifetime Data Anal., 2017). To construct the final risk-stratification score, we used non-parametric clustering of CIR curves through k-medians algorithm (Villanueva N. et al. Stat. Med., 2019). Results We confirm the prognostic classifier NFRP in the NGS era and evaluate the impact of 39 new gene alterations in the adult cohort. Alterations affecting TP53, DNMT3A, IDH1/2, IKZF1, PI3K pathway ( PTEN, PIK3CA and PIK3R1), EP300, and PHF6 were independent prognostic factors in adult T-ALL. This led us to propose the first NGS-based classifier in T-ALL defining low risk patient (LR) as those with N/F, PHF6 or EP300 mutations without N-K-RAS, PI3K pathway , TP53, DNMT3A, IDH1/2 and IKZF1 alterations (234 of 440 patients, 53%). In the adult cohort, the NGS-based classifier separates a high-risk group (HR) (n=90/198, 45%) with a 5-year CIR of 47% (95%CI:36%-57%) and a low-risk group (LR) (n=108/198, 55%) with a 5-year CIR of 21% (95%CI:14%-29%) (p<0.0001). Our NGS-classifier was validated in the pediatric cohort, with a 5-year CIR of 35% (95%CI:26%-44%) in HR group (n=116/242, 48%) and 5-year CIR of 17% (95%CI:11%-24%) in the LR group (n=126/242, 52%) (p=0.001) ( Figure A). Since the NGS-based classifier is highly prognostic independently of minimal residual disease (MRD) at end of induction (cutoff 10 -4) and white blood cells count (WBC) (cutoff 100 x 10 9/L), we then developed and externally validated a global risk stratification model incorporating MRD1, WBC at diagnosis and the NGS-classifier. This model identifies 3 subgroups at CR1: a large favorable Risk (CR1-FAV) group (231/332, 70%) with CIR at 5 years estimated at 19% (95%CI:14%-24%) ( Figure B), a subgroup of Adverse risk (CR1-ADV) patients (30/332, 9%) with a 5-year CIR of 68% (95%CI:46%-82%) and an Intermediate risk (CR1-INT) group (71/332, 21%) with a 5-year CIR of 37% (95%CI:26%-48%). Conclusion T-ALL NGS-based stratification combined with WBC and MRD evaluation sharpens the prognostic classification in T-ALL and identifies a new subgroup of adverse risk patients who should benefit from innovative therapeutic approaches.

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.

Jmjd3 Drives Immature T-Cell Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 15% of pediatric and 25% of adult ALL cases. Despite an improved survival rate due to intensified chemotherapy and bone marrow transplantation, still 20% of T-ALL patients are primary refractory to the standard treatment or relapse, and have a poor clinical outcome. T-ALL patients can be classified into molecular genetic subgroups based on the expression or genomic alterations of specific oncogenic transcription factors. One such subgroup the early T-cell precursor ALL (ETP-ALL) is a heterogeneous subgroup of immature leukemias that often display high expression of LYL1 and LMO2. It's important to improve our knowledge of T-ALL biology and developing novel targeted anti-TALL therapies. We previously showed that the H3K27 demethylase Jmjd3 plays an important role in T-ALL and is a good candidate for the treatment of T-cell leukemia. Aim: Identify the role of Jmjd3 in the initiation of immature T-ALL. Methods: We generated a conditional Rosa26- Jmjd3 overexpression mouse model (Figure 1A), and crossed it with a hematopoietic-specific Vav-icre mice, to obtain Jmjd3 Vav mice. To investigate the role of Jmjd3 in tumor initiation, we monitored survival of a Jmjd3 Vav cohort. Arising tumors were characterized by immunohistochemistry, flow cytometry, and were transplanted into secondary immunodeficient recipients. To characterize the pre-leukemic compartment, we performed single cell feature barcoding and RNA sequencing. Also bulk RNAseq and proteomics were performed on primary and transplanted Jmjd3-driven tumors. To investigate the role of Jmjd3 in immature T-cell biology in human and mice, we analyzed thymi from 8-weeks-old control and Jmjd3 Vavmice, and performed OP9-DL4 stromal co-cultures with transduced CD34+ human cord blood cells that overexpressed JMJD3, respectively. We used ETP-ALL patient-derived xenografts (PDX) models to validate markers identified in single cell analysis. Results: We found that hematopoietic Jmjd3 overexpression resulted in hematological malignancies with 38% penetrance (n= 8/21) and a median survival of 63 weeks (Figure 1B). Immunohistochemistry confirmed overexpression of Jmjd3, decreased levels of H3K27me3, and increased levels of H3K27ac in Jmjd3 Vav tumors. Immunophenotypic analysis revealed immature Thy1.1 +CD4 -CD8 - cells in all obtained Jmjd3 Vav tumors (Figure 1C), indicative of an immature the early T-cell precursor (ETP)-like ALL. These immature T-ALLs gave rise to primary (7 weeks) and secondary (3 weeks) transplants upon their transplantation in immunocompromised mice. Bulk RNAseq confirmed that the transcriptional feature of the primary tumor were maintained upon transplantation. The role of Jmjd3 in stimulating immature thymocyte development was confirmed in thymi from 8-weeks-old Jmjd3 Vavmice and could be validated as well in human T-cell development using stromal OP9-DL4 co-cultures. Finally, we used single cell sequencing technologies to characterize both transcriptional features and cell-surface markers within the tumor fraction (Figure 1D). We confirmed that the tumor cluster was ETP-like with absence of CD1, CD5 and mature T-cell markers, and expression of CD44, Lyl1, and myeloid markers. Furthermore, we could identify cell-surface in immature Jmjd3-driven tumors that were also present in human T-cell stromal culture and ETP-ALL PDXs. Conclusion: We identified Jmjd3 as a driver of immature thymocyte populations that give rise to ETP-ALL.

The AKR1C3-Activated Prodrug, Achm-025, Eradicates Disease in Preclinical Models of Aggressive T-Cell Acute Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is exceptionally difficult to cure after relapse. T-ALL expresses significantly higher levels of the enzyme aldo-keto reductase family 1 member C3 (AKR1C3) compared with B-cell ALL. To exploit this finding, we developed a novel prodrug, ACHM-025, which is selectively activated by AKR1C3 to form a potent cell-entrapped DNA alkylating agent. ACHM-025 was designed to improve drug specificity and minimize toxicity observed with currently used DNA alkylating agents, such as cyclophosphamide (CPM), a prodrug which is activated systemically via liver enzymes. We evaluated the in vivo efficacy and AKR1C3 selectivity of ACHM-025 against a panel of 25 pediatric T-ALL patient-derived xenografts (PDXs) alongside standard-of-care therapy. Methods: AKR1C3 expression in T-ALL PDXs was determined by RNA-seq, immunoblotting and intracellular flow cytometry. For in vivo efficacy studies, PDXs were established as orthotopic models in immune-deficient NSG mice. Engraftment was assessed by enumerating the proportion of human versus mouse CD45 + cells (%huCD45 +) in the peripheral blood. Treatment commenced when the %huCD45 + reached ≥1% (Day 0), and events were defined as %huCD45 + ≥25% or leukemia-related morbidity. Drug efficacy was assessed by mouse event-free survival (EFS) and stringent objective response measures. For the single agent study, ACHM-025 (IP weekly, Days 0, 7, 14) was assessed against 25 T-ALL PDXs using a single mouse trial (SMT) format (one vehicle treated mouse, one drug treated mouse). For the consolidation therapy comparison, ACHM-025 (IP Days 0, 7) or CPM (IP Days 0, 7) combined with cytarabine (Ara-C; IP Days 0-4, 7-11) and 6-mercaptopurine (6MP; IP Days 0-4, 7-11) were assessed against a T-ALL PDX derived from a patient at relapse. For the relapsed/refractory (R/R) therapy study, ACHM-025 (IP Days 0, 7, 14) and nelarabine (IP Days 0-4, 14-18) were assessed against a T-ALL PDX derived from a patient at relapse. Results: ACHM-025 dose-limiting toxicity in cynomolgus monkeys was neutropenia, where the pharmacokinetic equivalent dose in NSG mice was well tolerated. We first evaluated the in vivo efficacy of ACHM-025 as a single agent using the SMT format across an extended panel of 25 T-ALL PDXs, to address the impact of genetic heterogeneity in pediatric ALL on drug response. Remarkably, 7/25 PDXs treated with ACHM-025 did not relapse over 250 days after the last treatment and a total of 22/25 T-ALL PDXs scored an objective response. In comparison, vehicle treated EFS ranged from 3-26 days for the 25 T-ALL PDXs. Importantly, AKR1C3 expression (mRNA, protein or intracellular) provides a predictive biomarker of efficacy, as ACHM-025 was significantly more effective against T-ALL PDXs with high AKR1C3 expression versus those with low AKR1C3 expression (p<0.0001). CPM is included in standard-of-care consolidation therapy for ALL in combination with Ara-C and 6MP. Comparing single agents, ACHM-025 was significantly more effective than CPM (T-C 25 vs. 57 days, p=0.0005, Table 1). The combination of ACHM-025, Ara-C and 6MP was significantly more effective than standard-of-care consolidation therapy (CPM, Ara-C and 6MP), more than doubling survival (T-C 36 vs. 75 days, p=0.0005, Table 1). Finally, we compared ACHM-025 with nelarabine, which is the only FDA approved agent for R/R T-ALL. Comparing single agents, ACHM-025 was substantially more effective than nelarabine (T-C 19 vs. 198 days, p=0.0005, Table 1). Importantly, relapsed disease remained sensitive to ACHM-025 re-treatment in vivo, with no evidence of acquired resistance. Remarkably, no mice treated with the ACHM-025/nelarabine combination relapsed over 250 days after the last treatment (Table 1). Conclusions: ACHM-025 exerted profound in vivo efficacy against T-ALL PDXs and eradicated the disease in 7 aggressive T-ALL PDXs. ACHM-025 was significantly more effective than CPM both as a single agent and when used in combination with Ara-C/6MP. Notably, ACHM-025 in combination with nelarabine was curative when used to treat a chemoresistant T-ALL PDX in vivo. The in vivo efficacy of ACHM-025 directly correlated with AKR1C3 expression levels, providing a predictive biomarker for response. These data provide strong preclinical evidence highlighting the potential of ACHM-025 as a targeted and effective therapy for aggressive forms of T-ALL.

Drug Response Profiling of Childhood T-Cell Acute Lymphoblastic Leukemia to Inform Functional Precision Medicine

Background/Aims: Despite the advancement in risk-directed, chemotherapy-based protocols, the prognosis of children with T-cell acute lymphoblastic leukemia (T-ALL) especially for those who were refractory to existing treatments or relapsed from remissions remains poor. The complexity of mutational landscape and lack of actionable genomic lesions collectively pose significant challenges for implementing targeted therapies based solely on genomics. To address this issue, this study seeks to utilize drug sensitivity and resistance test (DSRT) to directly assess the ex vivo response of primary T-lymphoblasts to a portfolio of targeted agents, aiming to establish its drug sensitivity profile and identify drug candidates of immediate clinical relevance to tailor functional precision medicine. Methods: A serum-free, mesenchymal stem cell (MSC)-based culture system was adopted to perform ex vivo DSRT on primary or patient-derived xenograft (PDX) samples of pediatric T-ALL. The latter was generated by transplanting NSG mice with paucicellular specimens with splenocytes collected at leukemia engraftment as the sources of patient-derived T-lymphoblasts. The 42-drug panel consisted of 35 targeted agents (1 in Phase I trials, 6 in Phase II trials, 6 in Phase III trials, 22 FDA-approved) and 7 standard chemotherapeutics. The DSRT tested six serial drug concentrations ranging from 0.1 to 10,000 nM. After 96 hours of co-culturing with MSCs, T lymphoblasts were identified by CD7 staining, while apoptotic cells were characterized by Annexin V/7-AAD. The half maximal inhibitory concentration (IC50) of individual drugs were curated from the dose-response curves. Unsupervised clustering was performed to map the overall drug response pattern. A case series of relapsed or refractory T-ALL were managed with targeted therapies guided by drug profiling results and evaluated for clinical responses. Results: DSRT was conducted on 19 primary and 14 PDX pediatric T-ALL samples from 20 patients. For primary samples, two had been tested for 42 drugs, ten were tested for 16 drugs and the rest underwent testing with varying numbers of drugs depending on cellularity and sample quality. All PDX samples were subjected to testing for 42 drugs. PDX samples retained the original pattern of drug response in matched patient materials (R 2 = 0.77-0.94, 8 paired samples). Unsupervised clustering identified 5 distinct groups of drug response: (i) highly active compounds with median IC50 <10 nM (4 drugs including the proteasome inhibitors bortezomib and carfizomib, BCL2 inhibitor navitoclax and HDAC inhibitor panobinostat); (ii) generally active compounds (median IC50 <230 nM, 10 drugs), including BCL2 inhibitors, MCL1 inhibitors, JAK2/3 inhibitors and MDM2 inhibitors; (iii) compounds with bimodal activities (wide IC50 ranges, 4 drugs), including dasatinib (tyrosine kinase inhibitor), sirolimus and everolimus (mTOR inhibitors) and standard chemotherapeutics; (iv) generally inactive drugs with sporadic exceptions (12 drugs); and (v) completely inactive compounds (12 drugs). Four children with T-ALL received treatment according to drug profiling results. Two patients were in first relapse, one in third relapse and one in a non-remission state. Three of these patients received bortezomib treatment, while the fourth patient received venetoclax. All patients responded to the treatments with complete or partial remission. One patient was bridged to allogeneic stem cell transplantation, and two were bridged to CD7-directed CAR therapies. Conclusions: Our study unveiled a pediatric-specific drug response profile for T-ALL and highlighted opportunistic drug candidates with clinical relevance. The successful implementation of functional precision medicine in our clinic elucidated its practicality and potential to enable personalized treatment for high-risk pediatric T-ALL.

Genetic and Functional Characterization of NUP98-Rearranged Leukemia

NUP98 rearrangements ( NUP98r) are recurrent alterations in pediatric acute myeloid leukemia (AML) and are associated with dismal outcomes (PMID:21813447). These structural variantsresult in fusion oncoproteins comprised of the N-terminus of NUP98 and the C-terminus of fusion partners. Among more than 30 fusion partners discovered so far, the associations between NUP98::NSD1 with myeloblastic AML and NUP98::KDM5A with acute megakaryocytic/erythroid leukemia (AMKL/AEL) have been appreciated, which often harbor distinct cooperating mutations (e.g., NUP98:: NSD1 with FLT3-ITD and WT1 mutations). In contrast, other fusions, such as NUP98:: RAP1GDS1 or NUP98:: HOXA9 can be found also in myelodysplastic syndrome (MDS) or T-cell acute lymphoblastic leukemia (T-ALL) (PMID:32766874). However, how these fusions and cooperating mutations contribute to the specific phenotypes of NUP98r leukemias is poorly understood. To investigate the genetic background of NUP98r leukemia, we collected RNA-seq, WGS, and WES data for 91 pediatric cases from St. Jude (n=71),AEIOP (n=3), and the TARGET study (PMID:29227476, n=17; Panel.A), which included AML (n=82), MDS (n=4), and T-ALL (n=5). NUP98::NSD1 (n=47, 51.6%) and NUP98::KDM5A (n=26, 28.6%) were the most common fusions, in line with previous observations. NUP98::RAP1GDS1 (n=6, 6.6%), an understudied NUP98 fusion, was the third most common fusion in this study. We also confirmed frequent mutations of WT1 and FLT3-ITD in NUP98::NSD1 AML and entire or partial chromosome 13 loss involving RB1 locus in NUP98::KDM5A AMKL, while we observed frequent mutations in RAS or epigenetics-related genes, CCND2/3, and RUNX1 preferentially in cases without alterations of WT1, FLT3 or RB1 loss. NUP98:: RAP1GDS1 and NOTCH1 mutations were strongly enriched in T-ALL. These findings suggest that fusions and cooperating alterations both contribute to the disease phenotypes. To study how NUP98 fusion oncoproteins (FOs) drive leukemogenesis and specific phenotypes, we applied Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to determine NUP98 FO binding for a primary sample (myeloblastic with NUP98::NSD1), CHRF-288-11 cell line (megakaryocytic with NUP98::KDM5A), and HA-tagged CD34 NUP98::KDM5A model (Panel.B-top). CUT&RUN using antibodies against the N-terminus NUP98 and histone modifications (H3K4me3, H3K27ac) revealed that these models shared NUP98 FO binding sites at the HOXA/B and RUNX1 loci, together with active histone marks. In contrast, binding patterns at differentiation-related gene loci were heterogenous. NUP98::NSD1 uniquely binds to CEBPA, which encodes a master regulator of myeloid differentiation, contrary to NUP98::KDM5A binding to GFI1B encoding regulator of erythromegakaryocytic differentiation . These data indicate that NUP98 FOs directly and uniquely regulate differentiation-related genes. We also performed CUT&RUN in AML cells with RUNX1::RUNX1T1 or UBTF tandem duplications, in which the N-terminus NUP98 antibody did not show any detectable peaks, confirming the specificity of the N-terminus NUP98 signals in NUP98r AML models. Experiments using a HA antibody in HA-tagged NUP98::KDM5A CD34 models confirmed that fusion oncoproteins are binding to these target regions. However, the C-terminus NUP98 antibody also detected similar signals as the N-NUP98 antibody, suggesting that wild-type NUP98 is also recruited to NUP98 FO target genes, possibly by a heteromer formation with NUP98 FOs and other NPC components. To understand the possible association between somatic mutations and the genomic binding of NUP98 FOs, we next investigated chromatin immunoprecipitation sequencing (ChIP-seq) data from the ENCODE study (PMID:22955991) for RB1, WT1, and STAT5 (downstream FLT3-ITD). Binding peaks of these proteins partly coincided with NUP98 peaks on representative target genes (Panel.B-bottom), suggesting that frequent mutations in NUP98r leukemia converge on the genomic interaction of NUP98 FOs. In conclusion, genomic characterization of NUP98r leukemia showed the association among fusion partners, cooperating mutations, and disease phenotypes. Epigenetic profiling revealed that NUP98 FOs directly regulate target genes in a fusion and differentiation-specific manner and provide a basis for further study to investigate how NUP98 FOs and cooperating partners drive leukemogenesis and disease phenotypes.

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.

Risk-Adjusted Therapies Yield Equivalent Outcomes for Adolescents and Young Adults (AYAs) Treated for Newly Diagnosed T-Cell Acute Lymphoblastic Leukemia (T-ALL) on Children's Oncology Group (COG) Studies AALL0434 and AALL1231

Introduction Previous clinical trials have reported that adolescent and young adult (AYA) patients treated for T-cell acute lymphoblastic leukemia (T-ALL) have lower disease-free survival (DFS) and overall survival (OS) and increased toxicity as compared to younger patients. Results for the AYA cohort treated on two COG international phase 3 trials for T-ALL are reported here. Patients and Methods AALL0434 enrolled 1562 T-ALL patients (2007-2014) and AALL1231 enrolled 615 T-ALL patients (2014-2019) treated on augmented BFM (ABFM) regimens. AYA patients were defined as age ≥16 years of age (AALL0434: n=226; AALL1231: n = 107). Both studies allowed enrollment up to age 31 years. On AALL0434, participants were randomized to receive escalating dose methotrexate (CMTX) without leucovorin rescue + pegaspargase or high dose MTX (HDMTX) + leucovorin rescue. Intermediate and high-risk patients were randomized to receive or not receive six 5-day courses of nelarabine (Nel). Risk group assignments in AYA vs non-AYA patients can be found in Table 1. On AALL1231, participants were randomized to receive or not receive bortezomib in Induction and Delayed Intensification. Key differences between AALL0434 and AALL1231 ABFM backbones included the use of prednisone in induction in AALL0434 versus dexamethasone in AALL1231, and cranial radiation therapy in the majority of AALL0434 participants versus only in patients with central nervous system involvement in AALL1231. Results On AALL0434, the 4-year DFS for AYA was 81% compared to 84% for those <16 years old (P=0.5965), and OS was 88% and 90% respectively (P=0.3478). AYA patients randomized to CMTX (n=70) had a superior DFS compared to those randomized to HDMTX (n=72) with 4-year DFS 96% versus 78% respectively (P=0.0038). No disadvantage was seen among AYA subjects who received nelarabine versus those who did not, as shown by a 4-year DFS 85% versus 89%, respectively (N=54) (P=0.8116), albeit the sample size was small. On AALL1231, the 4-year EFS for AYA (n=107) was 82.7±3.9% compared to 82.2 ±1.7% for non-AYA (n=508) (P=0.727), and OS was 86.5 ±3.5% and 88.4 ±1.5% respectively (P=0.553). No difference in outcome was seen for AYA patients randomized to receive/not receive bortezomib. Combined outcome data for AALL0434 and AAL1231 demonstrates 4-year EFS for AYA (n=333) of 82.2+2.4% vs 83.9+0.9% for non-AYA (n=1844) (P=0.52) (Figure 1) and OS of 87.5+2.1% vs 90+0.8% respectively (P=0.19). Significant differences in patient characteristics in AALL0434 were higher initial white blood counts (P=0.0004) and higher risk group category (P=0.0002) for AYA patients. There were no significant differences in the AALL0434 AYA cohort compared to younger patients in CNS disease status, race, ethnicity, remission status at end of induction, or day 29 end of induction minimal residual disease (MRD). Toxicity rates were not significantly different in non-AYA vs AYA patients with the exception of upper respiratory infection (9.4% vs 1.8%; P=0.0001) and osteonecrosis (4.8% vs 17.7%; P<0.0001) in AALL0434. Rates of sepsis, catheter-related infection, and other infections were not significantly different between AYA and younger patients in AALL0434. Analysis of patient characteristics and toxicity rates in AALL1231, as well as analysis of the combined toxicity data for AALL0434 and AAL1231, are currently underway and will be presented at the meeting. Conclusion COG AALL0434 and AALL1231 show outstanding overall outcomes for AYA patients with no significant difference in survival between AYA and non-AYA patients on either study, despite differences in the ABFM backbones and the higher risk features seen among the AYA group in AALL0434. There were similar occurrences of therapy-related toxicities for AYA and non-AYA study participants on AALL0434. Despite small numbers, CMTX holds a survival advantage for AYA T-ALL patients, with no survival disadvantage seen among AYA T-ALL patients who received Nel. Bortezomib does not offer significant benefit for AYA T-ALL patients.

Efficacy and Safety of AALL0232 Protocol in Adults with Acute Lymphoblastic Leukemia: Single Institution Experience

Background Over the past few decades, the outcomes for children with ALL have significantly improved with current regimens that yield a 5-year complete remission (CR) and event-free survival (EFS) rates of 98% and 70-80%, respectively. In contrast, the adult population has a CR of 85-90%, with a less satisfactory disease-free survival (DFS) rate of only 30-40% at 5-years. The feasibility of Children's Oncology Group (COG) AALL0232 protocol in older patients has not been established, especially adults older than 40 years and whether they will tolerate such unmodified pediatric based regimen. Therefore, we planned to assess our practice's outcomes in terms of the efficacy and safety of the COG AALL0232 protocol in adult ALL patients. Method This was a retrospective observational study that included patients ≥ 14 years with newly diagnosed ALL. Patients were excluded if they have secondary ALL. Our institute has adopted the AALL0232 protocol for both B-cell and T-cell ALL due to the similarities between the AALL0232 and CALGB 10403 protocols. MRD data was not included in our study since the cut-off for detection has been changed over the study period. Patient demographics and treatment-related outcomes will be reported using descriptive statistics. OS and RFS probabilities will be calculated using the Kaplan-Meier product-limit method from the date of pre-phase initiation. All P-values will be two-sided with P <0.05 indicating significance. Results Between 2014 and 2022, a total of 96 patients were included in our study. 74% had B-cell and 26% had T-cell ALL. The majority of the patients had standard risk ALL (23%). 65% of the patients were receiving AALL0232 protocol. Early death with AALL0232 protocol occurred in 11.1% of our population. The 2-year OS rate was 72.6% (95% CI 61.4-81.1) (Figure 1). The OS rate for B-cell ALL was 78.5% compared to 53.8% for T-cell ALL at 2-years (P =0.0912). The use of AALL0232 protocol was comparable to other protocols in terms of survival with a 2-year OS rate of 77% and 62.3%, respectively (P=0.0761). Adults with favorable cytogenetic risk had an OS rate of 100% compared to 67.6% for standard and 49.2% for poor risk (P =0.047). Adults younger than 40 years had a higher OS rate compared to those older than 40 (76.4% vs. 54.7%, P= 0.0187). RFS rate at 2-years was durable in 68.2% of the patients (95% CI 56.8-77.2) (Figure 2). RFS rates were 72.3% for B-cell and 54.8% for T-cell ALL. The AALL0232 Protocol had a significant impact on RFS compared to other protocols (73.6% vs. 56.5%, P= 0.0277) (Figure 3). Those who were younger than 40 had an RFS rate of 73.9%. The 2-year NRM occurred in 21.7% of the population. The AALL0232 protocol did not result in a higher NRM compared to other protocols (P= 0.492). Conclusion Although the use of AALL0232 protocol in the adult population has a remarkable impact on the rate of relapse, its effect on survival remains questionable especially for patients with T-cell ALL and poor cytogenetic risk. Another point of concern is the early death rate that was observed in our study. Further studies are needed to assess the overall impact of COG protocol in the adult population.

Survival Analysis of Adults with Acute Lymphoblastic Leukemia in Ecuador

Background Acute lymphoblastic leukemia (ALL) is the second most common acute leukemia in adults. Unfortunately, in Ecuador there are no survival studies in patients ≥15 years with ALL, thus, this is the first national report. The aim of the study was to describe clinical-demographic characteristics and overall survival (OS). Methods Medical records of 628 patients from 8 reference hospitals in Ecuador with acute lymphoblastic leukemia (ALL) aged ≥15 years, diagnosed between January/2015 and December/2022, were retrospectively reviewed. A high-risk group was defined as: age ≥ 35 years ; ≥ 30,000/mm3 leukocytes in B-cell acute lymphoblastic leukemia (B-ALL) or ≥ 100,000/mm3 leukocytes in T-cell acute lymphoblastic leukemia (ALL- T) ; CNS infiltration at diagnosis, and/or if EMR was not reached (<0.01%) negative at the end of induction. If none of these factors were present, it was classified as standard risk. OS and treatment-related mortality were evaluated. Before carrying out this study, the approval of a national research ethics committee was obtained, as well as the approval of the 8 participating centers. Results 628 patients with ALL were included in the study. Summary of clinical and demographic data Table 1. In this cohort of patients, 9 different regimens of intensive chemotherapy were used. 454/517 (87.8%) patients with B-ALL were considered high risk. 595/628 (94.5%) patients received intensive chemotherapy. 150/595 (25.2%) and 54/595 (9.0%) patients died during the induction and consolidation phase respectively, reaching a mortality related to the treatment of 34.2%. In 290/401 (72.3%) with B-ALL achieved a complete response (CR) after induction, also 199/401 (49.1%) patients achieved minimal residual disease (MRD) negative (<0.01%). 16/235 (6.8%) patients with B-ALL considered high risk who achieved CR received allogeneic hematopoietic stem cell transplantation (HSCT). 32/35 (91.4%) patients with T-cell acute lymphoblastic leukemia (T-ALL) received intensive chemotherapy, of which 17/24 (70.8%) achieved CR, MRD negative (<0.01%), in 8/20 (40.0%) and 3/17 (17.6%) who achieved CR received HCT. The median OS was 12 months (95% CI 9.8-14.1) with a 5-year OS of 25.6%. The median OS for intensive chemotherapy patients with B-ALL was 12 months (95% CI 9.8 - 14.1) with a 5-year OS of 25.8% and 8 months (95% CI 5.9 - 10.0) with a 5-year OS of 23.5% for T-ALL. The 5-year OS for patients with HSCT was 78.4% (median survival not reached). The median OS for patients with B-ALL who received intensive high-risk chemotherapy was 12 months (95% CI 9.7-14.2) while median survival was not reached in the standard-risk group (p=<0.01). Figure 1. In patients with ALL-B, regimens with or without L-asparaginase achieved a 5-year OS of 26.4% vs 22.9% (p=0.588), respectively. In multivariate analysis for patients with ALL-B on intensive chemotherapy, age [HR 1.02 (95% CI 1.00 - 1.04) p=0.014] was significantly associated with OS, as well as those patients who did not achieve negative an MRD [HR 1.82 (95% CI 1.23-2.69) p=<0.01]. Otherwise, the adolescent and young adults (AYA) group, BCR:ABL fusion, CNS infiltration and leukocyte number at debut, L-asparaginase chemotherapy regimens, and being classified as high-risk group did not show statistical differences associated with OS . Conclusion The reported OS rates in this cohort are considerably low when compared to with international available data, being mainly negatively affected by the high treatment-related mortality rate, and the small number of patients who reach a HSCT.

Genetic and Pharmacological Interruption of the SIX1/EYA2 Axis Impairs Leukemia Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

CD38 KO/CD38-CAR Human Primary Natural Killer Cells Enhance Cytotoxicity Against CD38-Expressing Primary Lymphoma, Leukemia, and Myeloma

Natural killer (NK) cells have strong cancer immunotherapeutic potential that can be further augmented by engineering with a chimeric antigen receptor (CAR). CD38, a transmembrane glycoprotein involved in signal transduction and cellular adhesion, is highly expressed on the surface of the hematologic malignancies multiple myeloma (MM), acute myeloid leukemia (AML), Burkitt Lymphoma (BL) and T-cell acute lymphoblastic leukemia (T-ALL). FDA approved CD38 monoclonal antibodies (mAb) daratumumab and isatuximab are used to treat MM in patients, and have been tested to treat other CD38 expressing malignancies. NK cells play an imperative role in the efficacy of these antibodies and have natural antitumor activity against these cancers. However, because NK cells also highly express CD38, anti-CD38 mAb can cause NK cell fratricide, limiting their efficacy. Here, we generated multiple CD38 scFv based on isatuximab sequences and generated fratricide-resistant primary CD38-CAR NK cells using CRISPR/Cas9 genome editing and AAV6 gene delivery. Using this approach, we inserted the CD38-CAR gene into the CD38 locus to efficiently generate simultaneous CD38 KO and CD38-CAR KI with a mean expression level of %60. The CAR-expressing NK cells could be expanded on mbIL21-expressing feeder cells to a clinically relevant number of cells with no decline in CAR-expression level during two weeks of expansion confirming the lack of CAR mediated fratricide. We evaluated transduction efficiency and expression of CD38-CAR, and compared the cytotoxic function of two constructs with CD8 transmembrane (TM)/stalk and 41BB signaling domains and reverse heavy and light chains against AML, MM, T-ALL and BL immortalized cell lines and primary patient samples. We enhanced CD38 expression in target cancer cells by pre-treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A that pushes cells to maturation, consequently upregulating CD38. The sequential treatment of target cells with 10nM ATRA for 48 hours enhanced the cytotoxic activity of the CD38 KO/CD38-CAR-NK cells against primary MM, BL and AML samples. This would be particularly important for patients with low expression of CD38 on their cancers. The enhanced anti-tumor activity of the CAR was also studied and observed by CyTOF. Additionally, to better understand the therapeutic potential of CD38-CAR NK cells compared to CD38-CAR T cells we generated CD38 KO/CD38-CAR T cells using the same engineering approach and comparing their cytotoxic function. We showed that both CD38-CAR-T and CD38-CAR-NK cells have comparable anti-tumor activity compare to their paired non-edited wild type cells. Finally, we investigated the need for a CD38 KO to develop a fratricide-resistant therapy by comparing the cytolytic and metabolic functions of CD38 +/AAVS1 KO and CD38 KO CD38-CAR NK cells. We conclusively report a CD38-CAR NK cell therapy with enhanced metabolism and cytotoxicity toward a variety of hematologic malignancies, which can be further augmented by combination treatment with ATRA to target CD38-low malignancies.

Gene-Edited Universal CD7 Chimeric Antigen Receptor T Cells for Pediatric T-Cell Acute Lymphoblastic Leukemia or Lymphoblastic Lymphoma

Background: T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are highly aggressive T-lineage malignancies characterized by an infiltration of immature T cells in the bone marrow (BM) and peripheral blood (PB) or extramedullary organ involvements. Effective treatments were elusive, and the long-term survival of patients with refractory/relapsed (R/R) T-ALL/LBL remains poor despite allogeneic hematopoietic stem cell transplantation (allo-HSCT). CAR-T therapies for T-cell malignancies remain underdeveloped due to the shared antigenicity between normal and malignant T cells. CD7 is highly expressed on the surface of T-ALL/LBL T cells and is considered a viable CAR-T therapeutic target. However, CD7-targeting immunotherapies may be compromised by T-cell fratricide and extermination. And one of the biggest challenges with autologous CAR-T cell therapy is to isolate a sufficient number of healthy T cells from malignant T cells. Thus, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR9-associated protein 9 (Cas9)-mediated gene ablation has been used to produce universal CD7-targeted CAR T cells (UCD7-CAR) from suitable healthy donors that no longer express CD7 and TCR. In vitro killing tests, animal tests, and phase 1 clinical trials have preliminarily verified the efficacy and safety of universal CD7 CAR-T cells. Here we investigated the safety and efficacy of the universal CD7 CAR-T therapy in phase 2 clinical trial(NCT05454241) for pediatric R/R T-ALL/LBL patients. Aims: To investigate the efficacy and safety of the universal CD7 CAR-T therapy for pediatric R/R T-ALL/LBL patients. Methods: Patients over three years of age whose disease failed ≥ 2 lines of therapies and had ≥ 80% CD7 expression were eligible, including those who relapsed within 12 months after complete response or relapsed more than 12 months after complete response and failed to achieve remission induced by one or more courses of standard treatment; and patients with recurrence after hematopoietic stem cell transplantation. A lymphodepletion regimen was administered to all patients using fludarabine (30 mg/m2 per day) and cyclophosphamide (500 mg/m2 per day) for three consecutive days (day -5 to day -3) before CAR-T infusion. Patients received one single infusion of UCD7-CAR T cells at a median dose of 2.0×10^6/kg. Consolidative allo-HSCT after remission was allowed after day 28 after UCD7-CART infusion at the discretion of the physician and patient preference. All surviving patients were followed until July 15, 2023; the median follow-up was 109 days (54 to 117 days). Results: Five eligible children, 1 with r/r T-LBL and 4 with r/r T-ALL, were enrolled between December 2022 and April 2023 (Table 1). The median age of the patients was 5 (4-11) years. One girl(Patient 04) had a history of allo-HSCT before enrollment. Patients were heavily pretreated with a median of 3(2-6) lines of therapies. At registration, the median proportion of blasts in 4 T-ALL patients was 47.275% (6.0%-56.0%) in BM by FCM along with CNS(n=2) or testis(n=1) involvement. In contrast, 1 T-LBL patient(patient 02) did not have BM blasts but with bulky mediastinal masses ≥7 cm in diameter. All five patients had no or mild CRS (no CRS, n=1; grade 1, n=3; grade 2, n=1), and most(n=4) did not show symptoms of neurotoxicity. Patient 04, who received a prior allo-HSCT approximately seven months before CAR-T therapy, had a Herpes virus 6 (HHV-6B) encephalitis on day 17 after CAR-T infusion, but no GVHD was observed. On day 28, 4 of 5 (80.0%) patients who had BM involvement achieved MRD-negative complete remission (CR; n=2) or CR with incomplete hematologic recovery (CRi; n=2); but patient 02 with bulky mediastinal masses did not have an obvious response. Until the cutoff date, patient 04 still kept a sustained MRD negative CR; patient 05 relapsed again 49 days after CAR-T infusion, and the other three patients received a subsequent allo-UCBT with a CR state. All the children are still alive. Conclusion:UCD7-CAR T therapy is a safe and highly effective pediatric T-ALL/LBL treatment. More patients and longer follow-ups are needed for validation.

Prognostic Factors Impacting Post-Transplant Outcomes in Adult T-Cell Acute Lymphoblastic Leukemia: A Registry-Based Study By the EBMT Acute Leukemia Working Party

Background: T-cell acute lymphoblastic leukemia (T-ALL) predominantly affects individuals in late childhood and young adulthood and historically had high mortality rates. The outcome of Pediatric inspired chemo protocols in the T-ALL have improved in the past years due to the advancement in treatment and supportive care but the data is less mature, although adult patients still face challenges. Limited studies have directly explored the impact of patient- and transplant-related factors on post-transplant outcomes in adult T-ALL patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: A retrospective registry-based analysis was conducted across multiple centers using data from the European Society for Blood and Marrow Transplantation (EBMT) registry. Inclusion criteria were adult T-ALL patients (>18 years old) who underwent their first allo-HSCT in first complete remission from matched sibling donors (MSD), unrelated donors (UD) (10/10 or 9/10) or haploidentical donors between 2010 and 2021. Cord bloods were excluded. Multivariable Cox regression analysis was performed to examine the associations between patient/transplant characteristics and outcomes. Results: A total of 1907 patients were included in the study. The median age at transplant was 33.4 years (18.1-75). The median follow up was 2.9 years (2.6-3.1), and the median year of HSCT was 2016 (2010-2021). Patients were 70 % male with 67% being CMV-positive, at the time of transplant. Donors were predominantly male (64%), and (23%) of transplants involved female donors to male patients. The median period between diagnosis and HSCT was 5.9 months (0 -23.8). Most transplants were from matched sibling donors (MSD) (45%) followed by UD (43%) and 12% were from Haploidentical transplants. Most patients underwent myeloablative conditioning with total body irradiation (TBI)-based regimens (69%) and most patients received peripheral blood stem cells (84%). Cyclosporine with methotrexate was the most common graft-versus-host-disease (GVHD) prophylaxis (54 %). In vivo T cell depletion was used in 44%. The 2-year overall survival (OS) was 69.4%, and leukemia -free survival (LFS) was 62.1%. The cumulative incidence of acute GVHD (aGVHD) grades II-IV at 100 days was 32.6%, and grades III-IV was 10%. The cumulative incidence of chronic GVHD (cGVHD) at 2 years was 37.3%, and extensive cGVHD was 16.8%. Multivariate analysis yielded significant associations between patient/transplant characteristics and outcomes. Advanced age at transplant was associated with poorer outcomes, including LFS (HR=1.11, p=0.004), GVHD-free, relapse-free survival (GRFS) (HR=1.06, p=0.04), OS (HR=1.12, p=0.002), and higher non-relapse mortality (NRM) (HR=1.23, p=0.001); HR for 10y increment. A later year of HSCT was associated with improved GFRS (HR=0.89, p=0.001), OS (HR=0.9, p=0.02), and decreased NRM (HR=0.82, p=0.008), aGVHD II-IV (HR=0.83, p=0.001), and cGVHD (HR=0.8, p=0.001); HR for 3y increment. TBI was specifically superior with improved LFS (HR=0.79, p=0.02), GRFS (HR=0.83, p=0.04), and lower relapse incidence (RI) (HR=0.65, p=0.001). Female-to-male transplant combination had a negative impact on GFRS (HR=1.21, p=0.02), OS (HR=1.23, p=0.048), and on the risk of cGVHD in general (HR=1.39, p=0.002) and specifically extensive cGVHD (HR=1.47, p=0.01). The use of in vivo T-cell depletion had significant benefits in terms of GFRS, aGVHD grade III-IV, cGVHD, and extensive cGVHD. Conclusion: This large study identified prognostic factors, such as age at transplant, donor type, and conditioning regimen, in influencing key outcomes including OS, LFS, GVHD incidence, and NRM in adult T-ALL patients undergoing allo-HSCT. Importantly, a significant improvement over time in post-transplant outcomes was noted. These findings hold great promise for new adapted treatment strategies and can serve as a benchmark for future studies in that setting.

Selinexor Synergizes CX4945 on Anti-Tumor Effect By Targeting XPO1/Ikros/c-Myc Signaling in T-Cell Acute Lymphoblastic Leukemia

Background Acute lymphocytic leukemia (ALL) is an aggressive hematologic malignancy with poor outcomes. The IKZF1 (Ikaros) gene plays a crucial role in normal hematopoiesis as well as acts as a leukemia suppressor in ALL. Casein kinase II (CK2) inhibitor, CX4945, has shown potential function in restoring Ikaros on transcriptional repression of target genes in ALL. Exportin-1 (XPO1) is a critical mediator of nuclear export, which is essential for regulating the cell cycle and proliferation of normal and malignant tissues. Increased expression of XPO1 has been observed in various types of cancer, including ALL. In this study, we investigated the combined effects of Selinexor and CX4945 in T-cell ALL (T-ALL) and explored the underlying mechanisms. Methods Cell Counting Kit-8(CCK-8) was used for cell viability assay, and Annexin-V/PI staining following flow cytometry analysis for apoptosis. qPCR was used for gene expression in CEM and Molt4 T-ALL cell lines. CalcuSyn analysis was performed for a synergistic effect. Results Treatment of CX4945 or Selinexor significantly led to cell proliferation arrest of CEM and Molt4 cells in a dose-dependent manner. The combination of Selinexor with CX4945 significantly enhanced the cell proliferation arrest of CEM and Molt4 cells versus the single drug controls, and CalcuSyn analysis exhibited a synergistic effect of the two drugs in the cells (Fig.1A, B). The combination also significantly increased the apoptotic cells compared to the single-drug controls (Fig.1C). c-Myc oncogene plays an essential role in human cancers. Our results showed that c-Myc mRNA level was significantly higher expressed in the cohort of T-ALL patients compared to that of normal bone marrow controls (Fig.1D). To gain insight into the underlying mechanism of the synergy, we observed the effect of the drugs on the expression of c-Myc. Results showed that the combination of Selinexor with CX4945 significantly down-regulated the expression of c-Myc compared to the single-drug controls in CEM and Molt4 cells (Fig.1E). Moreover, the combination of Selinexor with CX4945 significantly enhanced the cell growth arrest in primary cells from T-ALL patients compared to single drug controls (Fig.1F). Taken together, our data indicated the synergistic effect of Selinexor with CX4945 on cell proliferation arrest and apoptosis by down-regulation of c-Myc expression. We previously reported that Ikaros directly regulates c-Myc transcription in ALL and CX4945 suppresses c-Myc expression in a Ikaros-dependent manner. Also, c-Myc is the cargo of XPO1. Thus, our results in this study reveal a model that treatment with XPO1 inhibitor, Selinexor prevents the nuclear transport of c-Myc and leads to its nuclear accumulation and down-regulation of c-Myc expression. The combination of Selinexor and CX4945 showed the synergistic anti-tumor effect by suppressing c-Myc expression in both transcriptional and post-transcriptional levels. The mechanism model is summarized in Fig.1G. ConclusionsThe combination of Selinexor and CX4945 has synergistic effects on cell proliferation arrest and apoptosis in T-ALL by targeting the XPO1/IKROS/c-Myc signaling. Our results also provide experimental evidence for the new combination of Selinexor and CX4945 as a new potential therapeutic option for T-ALL patients.

Picalm/MLLT10 Expression in Zebrafish Is Associated with an Increase of Hematopoietic Stem Cells and Leads to Leukemia

The PICALM/ MLLT10 (C/A)fusion gene is the result of the (10;11)(p13;q14) translocation, and is found in about 1% of all AML patients, 30% of T-cell ALL with a TCR γ/∂ rearrangement and in some patients with lymphoma. AML patients with a t(10;11)(p13;q14) have poor long-term survival. Mouse models of t(10;11)(p13;q14) show that C/A and the C/A minimal fusion (CA/MF) are strong oncogenic drivers, and that C/A must be expressed in the hematopoietic stem cells (HSCs) to cause leukemia. Patient samples and mouse models of C/A leukemia show that these leukemias harbor additional, cooperating mutations. Zebrafish (ZF) have become an attractive animal model for studying hematological disorders. ZF can be easily genetically modified and kept in large numbers. Although T cell ALL models have been successful in ZF, AML models have not been as successful. Most ZF AML models fail to recapitulate the phenotype, especially transplantability. Here we report a C/A transgenic zebrafish AML model (R:CA/MF). We used the murine Runx1 promoter to direct CA/MF expression to zebrafish HSCs using Tol2-mediated transgenesis. We detected the expression of the CA/MF transcript using RT-PCR in 3 dpf (days post fertilisation) heart marker positive transgenic F0 embryos and the expression of FLAG-tagged CA/MF protein with whole mount in situ immuno histochemistry in stable transgenic R:CA/MF embryos at 3 dpf. We performed whole mount in situ hybridisation (WMISH) and qPCR for hematopoietic genes to examine the impact of CA/MF on zebrafish hematopoiesis. Based on qPCR analysis, we observed that the expression of pu1 (p value <0.0120) and gata1 (p value <0.0004) was significantly decreased in 3 dpf R:CA/MF embryos compared to WT, whereas the expression of mpx at 2 dpf (p value <0.0132) and 3 dpf (p value <0.0378) was significantly increased. Our WMISH analysis showed that the expression of lyz was significantly decreased in R:CA/MF compared to WT embryos at 2 dpf. To investigate the effect of CA/MF expression on HSCs, we injected the R:CA/MF construct into fertilised oocytes of R:GFP;kdrl:RFP fish (R:GFP marks HSCs and Kdrl:RFP marks blood vessels). The results from intravital confocal microscopy showed that the 3 dpf R:GFP larvae that had been injected with the R:CA/MF vector had about a 2.5 fold increase (p value <0.0001) of the GFP expressing cells (HSCs) in both the caudal hematopoietic tissue and circulation compared to control (R:Gal4) and un-injected larvae. These results were also confirmed by flow cytometry. CA/MF transgenic zebrafish developed an aggressive hematological malignancy with a latency of about 1 year and 60 to 70% penetrance. 68% of F1 R:CA/MF (15/22), 64% of F2 R:CA/MF (45/70), 62% of F3 R:CA/MF (22/35) and 62% of F4 R:CA/MF (25/40) adult ZF were euthanised or found dead. The median survival for F1 CA/MF transgenic ZF was 65.6 weeks and 69, 78.4, and 79.1 weeks for F2, F3, and F4, respectively. Histology analysis of R:CA/MF leukemic fish showed hypercellularity in kidney, spleen, heart, gills and eyes. Myeloperoxidase (MPO) staining showed that the number of MPO positive cells in kidney marrow was about 50% higher (p value <0.0001) in R:CA/MF compared to WT. Peripheral blood film analysis of R:CA/MF showed the presence of blast-like cells. In addition, R:CA/MF malignancies were associated with an expansion of either the precursor compartment (N=20/31), the myeloid compartment (N=8/31) or the lymphoid compartment (N=3/31) on forward/side scatter flow cytometry analysis. Transplantation assays showed that R:CA/MF leukemias were serially transplantable. The latency to leukemia development was about six weeks in the first, second, and third recipient fish, which was much shorter than the latency for the primary R:CA/MF leukemias. Our preliminary whole-exome sequencing (WES) analysis identified somatically mutated genes in CA/MF zebrafish leukemia with some of these genes having human orthologues that are known to be somatically mutated in human leukemia suggesting that these are cooperating driver mutations. Preliminary RNA-Seq experiments revealed that genes in the KRAS and MTOR pathways had increased expression in the CA/MF ZF leukemia. R:CA/MF leukemia model is a valuable model for understanding the molecular mechanisms of malignant cell transformation of C/A. It allows us to investigate the effects of C/A on HSCs in vivo and to study the fully developed leukemia via serial transplantation, WES, and RNA-Seq.

REM-422, a Potent, Selective, Oral Small Molecule mRNA Degrader of the MYB Oncogene, Demonstrates Anti-Tumor Activity in Mouse Xenograft Models of AML

The c-MYB (MYB) oncogenic transcription factor is a key regulator of hematopoietic cell differentiation and proliferation. Recurrent genetic lesions or dysregulation of MYB have been identified in a variety of cancers, including adenoid cystic carcinoma (ACC), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), pediatric low-grade glioma (pLGG), and others. AML is characterized by multiple oncogenic abnormalities and high MYB mRNA expression. Evidence from in vitro and in vivo animal studies suggests that MYB may be a key downstream effector of these oncogenic abnormalities through the direct regulation of genes such as BCL2, MYC and FLT3. Functional genomics studies reveal MYB dependencies in human leukemia cell lines and mouse models of leukemia. Together, these data suggest that MYB is an attractive therapeutic target for AML and other hematological malignancies. Therapeutic agents that directly target MYB have yet to be developed, presumably due to the challenges of targeting this intrinsically disordered transcription factor, which lacks tractable ligand binding pockets. Here, we describe the mechanism of action and biological activity of REM-422, a potent, selective, oral small molecule messenger RNA (mRNA) degrader of MYB. REM-422 was identified through screening and medicinal chemistry efforts directed towards identifying small molecules that induce the inclusion of a normally unused ‘poison exon’ (PE), which contains a premature termination codon, into the MYB pre-mRNA transcript. Poison exon inclusion activates the nonsense-mediated decay pathway and promotes the degradation of MYB mRNA. We demonstrate in a cell-free assay that REM-422 acts by selectively facilitating the binding of the U1 snRNP spliceosome complex, which governs exon usage by recognizing 5‘ splice sites, to oligonucleotides containing the MYB PE 5‘ splice site sequence. REM-422 treatment of human leukemia cell lines promotes poison exon inclusion, leading to degradation of MYB mRNA and subsequent reduction in MYB protein levels (Figure 1A). In a panel of AML cell lines, REM-422 demonstrated preferential anti-proliferative activity in cell lines with high MYB expression, and in those shown to be MYB-dependent through functional genomics studies. To understand the pharmacokinetic (PK) and pharmacodynamic (PD) effects of MYB in vivo, Kasumi-1 AML cells were implanted subcutaneously in CB17 scid immunocompromised mice and REM-422 was dosed by oral gavage after the formation of established tumors. QD PO administration of REM-422 demonstrated dose-dependent effects in tumors on MYB mRNA levels, with induction of the MYB poison exon leading to reductions of MYB functional mRNA. In a 21-day study, REM-422 demonstrated significant anti-tumor activity, including tumor regressions at well tolerated dose levels (Figure 1 left). To further investigate the activity of REM-422, AML patient cells were injected into NOG immunocompromised mice and allowed to engraft. After successful engraftment, mice were dosed by oral gavage once daily with REM-422 at 10 mg/kg or vehicle for 24 days. Flow cytometry analysis of bone marrow and peripheral blood samples demonstrated that REM-422 led to the eradication of human leukemia cells from both compartments (Figure 1 right). Together, these data demonstrate that REM-422 treatment leads to degradation of MYB mRNA in preclinical models of AML and supports the therapeutic potential for REM-422 in AML patients.

Comparative Analysis of Immune Effector Cell-Associated Hematotoxicity (ICAHT) and Infection Incidence Following CD19-Targeted CAR-T Therapy Versus CD7-Targeted CAR-T Therapy

Introduction CD19 CAR-T cell therapy has been revolutionizing the treatment of refractory /relapsed (R/R) B-cell acute lymphoblastic leukemia (B-ALL) over the past decade. While CD7 CAR-T therapy, an innovative approach, has only recently been applied over the last three years in our center to treat R/R T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL). While there are studies and guidelines from EBMT addressing hematologic toxicity, specifically Immune Effector Cell-Associated Hematotoxicity (ICAHT), following CD19 CAR-T treatment, the literature is still scarce on ICAHT occurrence post-CD7 CAR-T therapy. Here we conducted a retrospective study aiming at comparing ICAHT and infection incidences following the two distinct CAR-T therapies. Methods Data were extracted from two separate clinical trials. The CD19 CAR is structured with CD19 scFv, CD28-hinge, 4-1BB costimulatory region, and CD3ζ activation domains. T cells were activated by CD3/CD28 microbead 2 days before CD19-CAR lentivirus infection. “Naturally selected” anti-CD7 CAR-T cells (NS7CAR) were transduced an anti-CD7 CAR into bulk peripheral T lymphocytes via a lentivirus vector. NS7CAR is a 2nd generation murine-based CAR-T containing 4-1BB and CD3ζ co-stimulatory domains. All patients received lymphodepletion regimens of fludarabine (30 mg/m 2/d) and cyclophosphamide (300 mg/m 2/d) from Day -5 to Day -3 before CAR-T infusion. A single infusion of either CD19 CAR-T or CD7 CAR-T cells was administrated at a medium dose of 1×10 6/kg. Fitted patients undergo consolidation allogeneic hematopoietic stem cell transplantation (Allo-HSCT) within 1-3 months after CAR-T cell infusion. The hematology toxicity and infection were analyzed during the period from CD19 CAR-T or CD7 CAR-T cell infusion to Allo-HSCT. Hematology toxicity is graded according to “ICAHT: EHA/EBMT Consumption Grading and Best Practice Recommendations” (Table 1). Infection diagnosis includes both etiological diagnosis and imaging diagnosis. Results Between June 1, 2019, and September 22, 2022, 60 patients with R/R B-ALL patients received CD19 CAR-T therapy (https://clinicaltrials.gov NCT04546893, and NCT04792593). Meanwhile, between December 1, 2020, and July 16, 2022, 60 patients with R/R T-ALL/LBL received CD7 CAR-T therapy (NCT04572308, NCT04916860). The CD19 CART group had 19 patients ≤ 14 years old, with a male/female gender ratio of 33:27. The CD7 CART group had 20 patients ≤ 14 years old, with a gender ratio of 49:11. The incidence of severe early ICAHT (Grade III+IV, day 0-30) from CD19 CAR-T cells infusion to allo-HSCT was observed to be 18.3%, which was lower than the incidence of the early ICAHT post CD7 CAR-T therapy at 33.3%, p =0.035. However, the incidence of severe late ICAHT (after day+30) following CD19 CAR-T and the CD7 CAR-T therapy was similar: 27.8% vs. 28.6%, with no statistical difference( p=0.57). There was no statistical difference between CD19 CAR-T and CD7 CAR-T therapy groups in the infection rates with 28.3%vs. 36.7%, p=0.33. In the CD19 CAR-T group, bacterial infections were noted in 10 patients: 6 with Gram-positive bacteria (1 Enterococcus, 1 Streptococcus agalactis, and 4 Staphylococcus), and 4 with Gram-negative bacteria (1 Pseudomonas aeruginosa, 2 Acinetobacter baumannii, and 1 Klebsiella pneumoniae). Viral infections were observed in 7 patients (EBV 2, CMV 2, BKV 3), and 1 had fungal infection. Infection sites included pulmonary infections (7 cases), sepsis (10 cases), and other areas (4 cases). Post-CD7-CAR-T, bacterial infections were observed in 9 cases, 7 with Gram-positive bacteria (6 Staphylococcus, and 1 Streptococcus bradycardia) and 2 with Gram-negative bacteria (1 Pseudomonas aeruginosa, and 1 Klebsiella pneumoniae). Six cases had viral infections (EBV 2, CMV 2, HHV1 type 1, parainfluenza virus 1), and 6 with fungal infections. Infection sites included pulmonary infection (12 cases), sepsis (10 cases), and other areas (5 cases). Conclusions Our study noted a slightly higher incidence of ICAHT within 30 days following CD7 CAR-T infusion compared to CD19 CAR-T therapy. However, there was no statistical difference in the incidence of ICAHT beyond the 30-day. There was no statistical difference for infection rate after CD19 CAR-T and CD7 CAR-T. In both groups, Gram-positive bacteria were primary responsible for bacterial infections.

Venetoclax Combined with Azacitidine Was Effective and Safe for Relapsed/Refractory T-Cell Acute Lymphoblastic Leukemia/ Lymphoblastic Lymphoma: Preliminary Results of a Phase 2, Multicenter Trial

Introduction: Relapsed/refractory T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (R/R T-ALL/LBL) is highly heterogeneous. The available salvage treatment options for relapsed patients showed remission rates of less than 50% and a 5-year overall survival (OS) rate of less than 25%. Venetoclax, a Bcl-2 inhibitor, in combination with azacitidine, has demonstrated a high remission rate in 5 patients with R/R T-ALL/LBL in our previous report. Here, we report the interim analysis of a phase 2 study (NCT05149378) for the treatment of venetoclax combined with azacitidine for R/R T-ALL/LBL. Methods: From November 2021 to May 2023, a total of 18 R/R T-ALL/LBL patients were enrolled in the study. Among them, 11 were male and 7 were female. These patients were from seven hospitals, including the central hospital of the First Affiliated Hospital of Soochow University. All patients received salvage treatment with venetoclax (orally, 100mg on day 1, 200mg on day 2, and 400mg on days 3-21) and azacitidine（75mg/m 2/d on days 1-7, subcutaneously). Bone marrow evaluation was performed after the blood cell count recovery or within 42 days since the initiation of the treatment. Results: Among the 18 patients enrolled, 8 were diagnosed with T-ALL, 7 with ETP-ALL, and 3 with T-LBL (Table). 5/18 patients were relapsed T-ALL/LBL, 13/18 patients were refractory to previous treatments. The median age of the patients was 39 years (range: 30-53). After 1-2 courses of venetoclax in combination with azacitidine regimen. 4 (22.2%) achieved CR, 1 patient (5.6%) achieved CRh, 9 (50.0%) patients achieved CRi and 2 (11.1%) achieved MLFS. The overall response rate was 88.9%. With a median follow-up time of 24.1 months (95% CI: 6.8-41.4), the estimated median OS was 24.1 months (95% CI: 6.8-41.4) (Figure a) and the median relapse-free survival was 14.0 (95% CI: 3.8-24.2) months (Figure b). After the treatment, 12/18 (66.7%) patients underwent allogeneic hematopoietic stem cell transplantation. Five patients experienced relapse between 6 to 19 months after the venetoclax and azacitidine treatment. Four patients died because of relapse after the regimen, two patients died because of COVID-19. Grade 3 or higher treatment-related hematologic AE included neutropenia (15/18, 83.3%), anemia (6/18, 33.3%), and thrombocytopenia (4/18, 22.2%). Treatment-related non-hematologic AE included infections (6/18, 33.3%. 4 patients developed pneumonia, 1 patient had perianal infection, 1 patient developed sepsis), elevation of ALT and AST (5/18, 27.8%), and renal function impairment (2/18, 11.1%). There was no other grade 3 or higher non-hematologic AE and no treatment-related death was observed. Conclusion: Our results showed that venetoclax in combination with azacitidine was an effective salvage regimen for R/R T-ALL/LBL patients. The benefit of this regimen for distinct cytogenetic groups in R/R T-ALL/LBL needs to be analyzed in more cases.

Boric Acid Affects Cell Proliferation, Apoptosis, and Oxidative Stress in ALL Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a type of acute lymphoblastic leukemia from early T-cell progenitors. Interest grows in creating less toxic agents and therapies for chemo-resistant T-ALL cancer. Recently, elemental boron has special properties useful in the creation of new drugs. Studies have revealed the cytotoxic properties of boric acid (BA) on cancer, but not fully understood. We aimed to investigate the effect of BA on cell proliferation, apoptosis, and oxidative stress in the Jurkat cells. The effects of BA on cell viability were determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay for 24-48-72 h. The impact of BA on apoptosis was analyzed by acridine orange/ethidium bromide. Expression of apoptosis regulatory genes (Bcl-2, Bax, Caspase-3-8-9) and apoptotic miRNA (miR-21) was used by real-time quantitative polymerase chain reaction (RT-qPCR). The total oxidant status (TOS), total antioxidant status (TAS), and the oxidative stress index (OSI) value were calculated for oxidative stress. We determined the cytotoxic activity of BA on Jurkat cells by using XTT and defined the IC50 concentration (802.7 μg/mL) of BA. The findings clearly show that BA inhibited Jurkat cell proliferation dose-dependently. BA induced apoptosis through downregulated anti-apoptotic genes, and upregulated pro-apoptotic genes. Additionally, we found that BA significantly reduced the expression of miR-21 (p<0.001). Our findings demonstrated that different doses of BA increased TAS levels while decreasing TOS levels in Jurkat cells. Our study suggests that BA might be potential anti-cancer agent candidate in ALL via inhibition of cell proliferation, induced apoptosis, and reducing the amounts of anti-oxidants in cells.