Outcomes for pediatric patients with refractory or relapsed T-cell acute lymphoblastic leukemia (T-ALL) are poor, underscoring the need for improved therapeutic strategies. CD38, a type II transmembrane glycoprotein, is a promising target in T-ALL, with clinical trials evaluating CD38-targeting immunotherapies in frontline and relapsed settings. However, the biological role of CD38 in T-ALL has not been systematically defined. We interrogated CD38 biology through multimodal profiling of pediatric T-ALL samples. Bulk RNA sequencing of 1,335 primary tumors revealed that CD38 expression varies across genomic and immunophenotypic subtypes in T-ALL. Flow cytometry of 150 primary samples and CITE-sequencing of 40 cases demonstrated broad surface expression of CD38. A transcription factor CRISPR-screen identified RUNX1, RUNX3, and TP53 as candidate positive regulators of CD38. Metabolomic profiling of cell lines further revealed disruption of the polyamine pathway following CD38 perturbation. Supporting this finding, co-targeting CD38 with difluoromethylornithine (DFMO), a polyamine metabolism disruptor, improved survival in preclinical models. Across transcriptomic datasets, including primary tumors, cell lines, and patient-derived xenograft models, IL32 expression consistently decreased following CD38 loss or negativity, supporting an association between CD38 and inflammatory signaling pathways. Additionally, CD38 and LCK expression were positively correlated across majority of genomic subtypes, implicating SRC kinase signaling. Consistent with this, daratumumab in cell lines increased LCK phosphorylation, and combination therapy with dasatinib improved survival compared to monotherapy. Collectively, these findings define previously unrecognized interactions between CD38 and targetable pathways and genes in T-ALL and identify rational combinatorial strategies to enhance CD38-directed therapies and reduce relapse risk.

Abstract Background Central nervous system (CNS) tumors are the most common pediatric solid tumors and a major cause of childhood cancer-related mortality. With the advent of molecular profiling, the World Health Organization (WHO) has increasingly incorporated genomic data into its diagnostic framework. However, clinical grade comprehensive genomic profiling data from large cohorts of unselected pediatric CNS patients remain limited. Methods We summarize genomic data from clinically validated DNA/RNA profiling of 574 CNS tumors from 532 pediatric patients at a single institution and assess the clinical utility of these findings. Results Tier 1/2 variants were identified in 94.1% of patients with 90.8% of patients yielding clinically impactful findings. These findings resulted in a diagnosis change in 4.2% of patients and refined the histologic diagnosis in 22%. Prognostically relevant alterations were identified in 10% of patients while potential therapeutic targets were identified in 38%. Additionally, serial tumor testing in 36 patients enabled assessment of tumor evolution and differentiation of recurrent/relapsed tumors from independent primary tumors. Furthermore, 23.9% of patients were suspected to have germline pathogenic/likely pathogenic variants, with 73.2% confirmed through germline testing, representing 11.3% of the cohort. Conclusions Our study provides a large, integrated clinical dataset of genomic alterations in pediatric CNS tumors. Our findings highlight the clinical significance of genomic profiling of pediatric CNS tumors and underscore the necessity of integrating genomic results with pathologic, radiologic, and clinical features to ensure accurate tumor diagnosis and facilitate personalized, risk-adapted patient management.

The BCL2 inhibitor venetoclax has therapeutic activity in several hematological malignancies. In acute myeloid leukemia (AML), venetoclax combined with hypomethylating agents is the standard of care for patients unfit for intensive chemotherapy, but intrinsic and acquired resistance are common. Loss of p53 function is strongly associated with venetoclax resistance, and adding venetoclax to 5-azacitidine provides no overall survival benefit in TP53-mutant AML. Other frequent mechanisms of venetoclax resistance in AML include FLT3 mutations, MCL-1 upregulation, and altered mitochondrial metabolism. Unfortunately, it has been challenging to develop agents that target these mechanisms directly and combinatorially. Here we report that pitavastatin, an inhibitor of HMG-CoA-reductase, promotes apoptosis and overcomes several venetoclax resistance mechanisms in human AML cells. At clinically achievable concentrations, pitavastatin treatment has potent cytotoxic activity in cells with mutations in TP53 or FLT3, including primary cells resistant to venetoclax and 5-azacitidine. The apoptotic mechanism involves p53-independent PUMA upregulation and reduced MCL-1 expression. Pitavastatin also suppresses mitochondrial gene expression, oxidative metabolism, and MYC/E2F-driven transcription. These results provide a mechanistic rationale for clinical trials adding pitavastatin to AML regimens to prevent or overcome venetoclax resistance with TP53 mutations and other high-risk features.

Supplementary Figure S1. Generation of a conditional knock-in mouse model for the Enl-T1 mutation. Supplementary Figure S2. Impact of Enl mutation on the peripheral blood and spleen. Supplementary Figure S3. Characterization of Enl-T1 allele expression and concurrent mutations in heterozygous knock-in Enl-T1 mouse model. Supplementary Figure S4. UBC-cre-ERT2/Enl flox-T1/+ mice develop aggressive acute leukemia following tamoxifen treatment. Supplementary Figure S5. Enl mutation leads to expansion of myeloid cells in mice in the leukemic phase. Supplementary Figure S6. Enl mutation leads to the decrease of B220+CD19+ B, CD4+T, CD8+ T cells in mice in the leukemic phase. Supplementary Figure S7. Impact of the Enl mutation on the bone marrow, peripheral blood, spleen, and thymus in mice in the pre-leukemic phase. Supplementary Figure S8. Enl mutation perturbs the normal hematopoietic hierarchy and leads to abnormal expansion of myeloid progenitors in mice in the leukemic phase. Supplementary Figure S9. Enl mutation does not lead to the expansion of myeloid progenitors in mice in the pre-leukemic phase. Supplementary Figure S10. Enl mutation promotes self-renewal properties of HSPCs. Supplementary Figure S11. Enl mutation-induced up- and down-regulated genes are related to distinct biological functions. Supplementary Figure S12. Enl mutation leads to a gain of myeloid differentiation signatures in HSPCs. Supplementary Figure S13. Mutant ENL-induced H3K27ac signals correlate with upregulation of development and inflammation associated transcriptional programs. Supplementary Figure S14. HSPCs gain H3K27me3 during differentiation in wildtype mice. Supplementary Figure S15. Differentiation-associated gain of H3K27me3 is impaired in Enl-mutated hematopoietic cells. Supplementary Figure S16. ENL mutants form condensate at key target genes and increase gene expression in HSPCs. Supplementary Figure S17. Condensate formation property correlates with mutant ENL’s oncogenic function in human CD34+ HSPCs. Supplementary Figure S18. Expression levels of different FLAG-ENL transgenes in LSK cells. Supplementary Figure S19. Condensate formation property correlates with mutant ENL’s oncogenic function in GMP cells. Supplementary Figure S20. Disrupting condensate formation by the H116P mutation reduces ENL-T1-induced increases in chromatin occupancy of FLAG-ENL, H3K27ac, and p300 at a subset of target genes. Supplementary Figure S21. Impact of mutant ENL on leukemia development and condensate formation. Supplementary Figure S22. Small molecule inhibition of the acetyl-binding activity of mutant ENL suppresses chromatin function and Hoxa cluster gene activation in LSK cells. Supplementary Figure S23. Small molecule inhibition of the acetyl-binding activity of mutant ENL impairs its chromatin and transcriptional function in HSPCs. Supplementary Figure S24. Small molecule inhibition of the acetyl-binding activity of mutant ENL inhibits its impact on the self-renewal property in HSPCs.

Supplementary Table S1. Genes differentially expressed between Enl-T1 and Enl-WT LSK cells. Supplementary Table S2. Genes differentially expressed between Enl-T1 and Enl-WT GMP cells. Supplementary Table S3. Genes differentially expressed between Enl-T1 and Enl-WT L-GMP cells. Supplementary Table S4. Genes differentially expressed between Enl-T1 and Enl-WT cKit + Mac1+ cells. Supplementary Table S5. Genes differentially expressed between Enl-T1 and Enl-WT cKit-Mac1+ cells. Supplementary Table S6. Shared Enl-T1 up-regulated DEGs in LSK, GMP, and L-GMP cells. Supplementary Table S7. Expression of Hoxa genes in Enl-WT and Enl-T1 hematopoietic populations. Supplementary Table S8. GSEA gene sets used in Supplementary Figure S12. Supplementary Table S9. GSVA score of patients from TARGET-AML database. Supplementary Table S10. H3K27ac peaks in all hematopoietic populations. Supplementary Table S11. H3K27ac differential regions of ENL-T1 versus ENL-WT in LSK cells. Supplementary Table S12. H3K27ac differential regions of ENL-T1 versus ENL-WT in GMP cells. Supplementary Table S13. H3K27ac differential regions of ENL-T1 versus ENL-WT in L-GMP cells. Supplementary Table S14. H3K27ac differential regions of ENL-T1 versus ENL-WT in cKit + Mac1+ cells. Supplementary Table S15. H3K27ac differential regions of ENL-T1 versus ENL-WT in cKit-Mac1+ cells. Supplementary Table S16. T1-UP DEGs associated with H3K27ac T1 gained DRs in all hematopoietic populations. Supplementary Table S17. H3K27ac T1 gained differential regions associated with T1-UP DEGs in all hematopoietic populations. Supplementary Table S18. p300 ChIP-seq normalized signal at T1 gained H3K27ac differential regions in Enl-WT and Enl-T1 GMP, L-GMP cells. Supplementary Table S19. T1 gained H3K27ac differential regions with both p300 UP and associated gene expression UP in Enl-T1 cells for GMP and L-GMP. Supplementary Table S20. T1 gained H3K27ac differential regions with p300 UP and associated gene expression UP in Enl-T1 cells for L-GMP under A-485 treatment. Supplementary Table S21. H3K27me3 peaks in wildtype hematopoietic populations. Supplementary Table S22. Hematopoietic differentiation associated-H3K27me3 peaks. Supplementary Table S23. H3K27me3 peaks in all hematopoietic populations. Supplementary Table S24. H3K27me3 differential regions of ENL-T1 versus ENL-WT in LSK cells. Supplementary Table S25. H3K27me3 differential regions of ENL-T1 versus ENL-WT in GMP cells. Supplementary Table S26. H3K27me3 differential regions of ENL-T1 versus ENL-WT in L-GMP cells. Supplementary Table S27. H3K27me3 differential regions of ENL-T1 versus ENL-WT in cKit + Mac1+ cells. Supplementary Table S28. H3K27me3 differential regions of ENL-T1 versus ENL-WT in cKit-Mac1+ cells. Supplementary Table S29. T1-UP DEGs associated with H3K27me3 lost differential regions in all hematopoietic populations. Supplementary Table S30. Group1 and group2 gene list in cKit + Mac1+ and cKit-Mac1+ cells. Supplementary Table S31. FLAG-ENL peaks in LSK cells expressing the indicated FLAG-ENL transgenes. Supplementary Table S32. FLAG-ENL gained regions of T1 versus WT in LSK cells expressing the indicated FLAG-ENL transgenes. Supplementary Table S33. H3K27ac T1 gained differential regions associated with T1 gained FLAG-ENL DRs in LSK cells expressing the indicated FLAG-ENL transgenes. Supplementary Table S34. Genes differentially expressed between Enl-T1-DMSO and Enl-WT-DMSO LSK cells. Supplementary Table S35. Genes differentially expressed between Enl-T1-DMSO and Enl-WT-DMSO in GMP cells. Supplementary Table S36. Expression of Hoxa genes under Enl-WT-DMSO, Enl-T1-DMSO and Enl-T1-TDI conditions in LSK and GMP cells. Supplementary Table S37. Oligos used in this study. Supplementary Table S38. Antibodies used in this study.

BACKGROUND:Relapse of B-cell acute lymphoblastic leukemia (B-ALL) with CD19-antigen loss after CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has a dismal prognosis. Novel immunotherapeutic strategies for this patient population are urgently needed. METHODS:We tested a novel, fully human anti-CD22/4-1BB CAR T-cell construct, CART22-65s, in parallel phase I studies for pediatric and adult B-ALL. After lymphodepletion, CART22-65s was infused using a 3-day fractionated dosing scheme, allowing for omission of the second and third doses in cases of early cytokine release syndrome (CRS). RESULTS:Twenty-two patients, all with relapse after prior CD19-directed immunotherapy, were enrolled. Of 19 infused patients (pediatric, n=17; adult, n=2), 14 (74%) achieved a complete remission (CR), including 4 of 6 (67%) patients refractory to prior inotuzumab. Five of 14 patients in a CR proceeded to consolidative hematopoietic cell transplantation (HCT). With a median follow-up of 38 months, the 12-month relapse-free survival rate was 38.4% (95% CI 19.3% to 76.5%) and overall survival rate was 52.6% (95% CI 34.3% to 80.6%). Two patients received additional CART22-65s treatments for subsequent CD22-positive relapses; one achieved another CR. All CRS (n=17, 89%) and neurotoxicity (n=4, 21%) events after initial infusion were grades 1-2. The only grade 3 CRS/neurotoxicity and the only high-grade immune effector cell-associated hemophagocytic lymphohistocytosis-like syndrome occurred in the retreatment setting. In vivo cellular kinetic data revealed robust CART22-65s proliferation by quantitative PCR peaking at a median of 20 days postinfusion, with the cells persisting out to month 42 in one patient who achieved a long-term remission with CART22-65s alone. CONCLUSIONS:The favorable safety profile and high remission rates in exceedingly refractory B-ALL support the continued development of CART22-65s but also highlight the need to use the product in combination with HCT or other novel strategies. TRIAL REGISTRATION NUMBERS:NCT02650414 and NCT03620058.

Unbiased kinome-wide CRISPR screening identified DYRK1A as a potential therapeutic target in KMT2A-rearranged (KMT2A-R) B-acute lymphoblastic leukemia (ALL). Mechanistically, we demonstrate that DYRK1A is regulated by the KMT2A fusion protein and affects cell proliferation by regulating MYC expression and ERK phosphorylation. We further observed that pharmacologic DYRK1A inhibition markedly reduced human KMT2A-R ALL cell proliferation in vitro and potently decreased leukemia proliferation in vivo in drug-treated patient-derived xenograft mouse models. DYRK1A inhibition induced expression of the proapoptotic factor BIM and reduced the expression of BCL-XL, consequently sensitizing KMT2A-R ALL cells to BCL2 inhibition. Dual inhibition of DYRK1A and BCL2 synergistically decreased KMT2A-R ALL cell survival in vitro and reduced leukemic burden in mice. Taken together, our data establishes DYRK1A as a novel therapeutic target in KMT2A-R ALL and credential dual inhibition of DYRK1A and BCL2 as an effective translational therapeutic strategy for this high-risk ALL subtype.

R code of our analysis clock like mutational signatures in Hodgkin lymphoma WGS and WES.

The BCL2 inhibitor venetoclax has therapeutic activity in several hematological malignancies. In acute myeloid leukemia (AML), venetoclax combined with hypomethylating agents is the standard of care for patients unfit for intensive chemotherapy, but intrinsic and acquired resistance are common. Loss of p53 function is strongly associated with venetoclax resistance, and adding venetoclax to 5-azacitidine provides no overall survival benefit in TP53-mutant AML. Other frequent mechanisms of venetoclax resistance in AML include FLT3 mutations, MCL-1 upregulation, and altered mitochondrial metabolism. Unfortunately, it has been challenging to develop agents that target these mechanisms directly and combinatorially. Here we report that pitavastatin, an inhibitor of HMG-CoA-reductase, promotes apoptosis and overcomes several venetoclax resistance mechanisms in human AML cells. At clinically achievable concentrations, pitavastatin treatment has potent cytotoxic activity in cells with mutations in TP53 or FLT3. The apoptotic mechanism involves p53-independent PUMA upregulation and reduced MCL-1 expression. Pitavastatin also suppresses mitochondrial gene expression and oxidative metabolism. The pro-apoptotic actions of pitavastatin depend on depletion of geranylgeranyl pyrophosphate (GGPP) and can be recapitulated by inhibiting GGPP synthase or geranylgeranyltransferase-1 enzymes. These results provide a mechanistic rationale for adding pitavastatin to AML regimens to prevent or overcome venetoclax resistance.

Acute myeloid leukemia with mutations in TP53 (TP53mut AML) is fatal with a median survival of 6 months. RNA sequencing on purified AML patient samples showed that TP53mut AML had higher expression of mevalonate pathway genes. Using novel, isogenic TP53mut AML cell lines and primary samples, we determined that TP53mut AML resistance to AML chemotherapy cytarabine (AraC) correlated with increased mevalonate pathway activity, a lower induction of reactive oxygen species (ROS), and a mitochondrial response with increased mitochondrial mass and oxidative phosphorylation. Pretreatment with the statin class of mevalonate pathway inhibitors reversed these effects and chemosensitized TP53mut AML. The geranylgeranyl pyrophosphate (GGPP) branch of the mevalonate pathway was required for TP53mut AML chemoresistance. In addition to its role in mitochondria biogenesis, we identified a novel function of GGPP in regulating glutathione for management of AraC-induced ROS. However, statins alone were inadequate to fully reverse chemoresistance in vivo and in a retrospective study of 364 TP53mut AML patients who received chemotherapy concurrently with a statin. Finally, we identified clinical settings and strategies to successfully target the mevalonate pathway, particularly to address the unmet need of TP53mut AML.

Background CD19-directed chimeric antigen receptor T-cell (CART19) therapy has transformed the treatment landscape for relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Yet relapse occurs in approximately 50% of recipients. These patients and subgroups of pediatric patients in first relapse, currently ineligible for commercial CART19, have very poor outcomes with current approaches, warranting investigation of alternative strategies. We previously investigated a humanized CART19 (huCART19) in a Phase I trial with encouraging responses in both CAR-naïve and CAR-exposed cohorts (Myers JCO 2021). Here we report outcomes from a Phase II clinical trial (NCT03792633) of huCART19 for high risk r/r B-ALL, including early bone marrow (BM) relapse, a subgroup with historically poor outcomes despite intensive therapy. Methods Patients aged 0-29 years (y) with CD19+ B-ALL were eligible in 2 cohorts: CAR-naïve patients with B-ALL that is refractory, in high risk first relapse, second or greater relapse, or relapsed after or ineligible for hematopoietic stem cell transplant (HSCT); CAR-exposed patients with poor response to prior cell therapy. Patients received huCART19 at a dose of 5x106 CAR T cells/kg (maximum 2.5x108) after lymphodepletion (LD) with fludarabine and cyclophosphamide. Response was assessed on day 28 by BM and cerebrospinal fluid morphology, minimal residual disease (MRD) by flow cytometry, and biologic response by B cell aplasia (BCA). The primary endpoint was event-free survival (EFS). Secondary endpoints included overall response rate (ORR), defined as rate of complete remission (CR) or CR with incomplete hematologic recovery (CRi) with BCA, and relapse-free survival (RFS). Data cutoff was July 1, 2025. Results Of 106 patients enrolled, 100 were infused with huCART19 from 3/2019-8/2023. The median age at infusion was 12y (range 1-29), 44% were female, 20% Hispanic, 5% Asian, 7% Black, and 6% had Trisomy 21. Prior therapy included HSCT in 21%, blinatumomab in 21% and inotuzumab in 15%. The CAR-naïve cohort (n=52) included 25 with first early BM relapse within 36m of diagnosis (12 <18m). On pre-infusion BM performed post-LD, 13/52 (25%) had >25% blasts, 25/52 (48%) <0.01%. Three patients were inevaluable for response: 1 deemed ineligible due to myeloid lineage switch on pre-infusion BM; 1 died on day 2; 1 lost to follow-up at day 28. By day 28, 46/49 (94%) were in CR/CRi, 45/46 MRD-negative, 1 MRD inevaluable. With a median follow up of 51m, EFS and RFS at 2y were 65% (95% CI 52-81%) and 70% (95% CI 57-86%), and at 4y, 57% (95% CI 42-76%) and 62% (95% CI 47-81%). Relapse occurred in 14/46 (30%), of which 7 (50%) were CD19-. Ten patients pursued alternative therapy in remission, 7 for loss of BCA and 3 for MRD recurrence (2 CD19+, 1 CD19-). Overall survival (OS) at 2y and 4y was 74% (95% CI 63-87%). In a subgroup analysis of CAR-naïve patients treated for early BM relapse, ORR was 21/23 (91%), 2y EFS was 39% (95% CI 21-72%), RFS 45% (95% CI 25-81%), and OS 57% (95% CI 40-81%). The CAR-exposed cohort (n=48) included 20 with post-CART19 relapse and 28 with early (<6m) loss of BCA without relapse. On pre-infusion BM, 5/48 (10%) had >25% blasts, 34/48 (71%) <0.01%. By day 28, 43/48 patients were in CR/CRi, 43/43 MRD-negative; 6/43 did not establish BCA resulting in an ORR of 37/48 (77%). With a median follow up of 43m, EFS and RFS at 2y were 53% (95% CI 39-71%) and 72% (95% CI 57-91%), and at 4y, 50% (95% CI 36-68%) and 68% (95% CI 52-88%). Relapse occurred in 9/37 (24%), of which 4 (44%) were CD19-. Eleven patients received alternative therapy in remission, 6 for loss of BCA, 4 for CD19+ MRD recurrence, and 1 for therapy-related myeloid neoplasm. OS at 2y and 4y was 77% (95% CI 66-90%). CRS was reported in 47/52 (90%, 10 grade [Gr] 3, 5 Gr 4 on Penn scale) CAR-naïve patients and 38/48 (79%, 1 Gr 3, 2 Gr 4) CAR-exposed. There was 1 death prior to day 28, due to gastrointestinal hemorrhage on day 2 in the setting of progressive ALL and Gr 2 CRS. CAR neurotoxicity was reported in 14/52 (27%, 2 Gr 3, 2 Gr 4) CAR-naïve patients and 7/48 (15%, 1 Gr 3, 1 Gr 4) CAR-exposed, with 1 case of Gr 3 cerebral edema, fully recovered, and 1 case of ongoing myelopathy. Conclusions HuCART19 produced durable remissions in high risk r/r B-ALL and demonstrated efficacy as salvage therapy for those with poor response to prior CAR therapy, comparing favorably to historical outcomes in this extremely high risk group.

We report a case of acute myeloid leukemia with megakaryoblastic differentiation (AMKL) that developed after an initial B-lymphoblastic leukemia (B-ALL) with low hypodiploidy. Although the AMKL was initially thought either to be a phenotypic change from the original B-ALL or to have arisen as a result of treatment (acute myeloid leukemia, post cytotoxic therapy, AML-pCT [WHO]; AML, therapy related [ICC]), genetic evaluation of both the AMKL and the B-ALL suggest that neither of these considerations was correct. Rather, the AMKL did not harbor the most common genetic hallmark of AML-pCT-rearrangement of KMT2- and was genetically distinct from the B-ALL. Both the B-ALL and the AMKL, however, showed an identical TP53 mutation by next generation sequencing (NGS), while germline testing was negative for this mutant allele. Hence, either the patient had a tissue restricted constitutional TP53 mutation or had a somatic mutation in a multipotent hematopoietic precursor. This case highlights the necessity for close monitoring of patients with TP53-mutant tumors, as they may develop multiple lesions despite negative germline testing.

Inhibitors of the menin-KMT2A interaction are promising agents for the treatment of KMT2A-rearranged leukemias. We evaluated menin inhibition in patient-derived xenografts of KMT2A-rearranged leukemias with high-risk features. Three acute myeloid leukemias with high-risk fusion partners (mixed-lineage leukemia-10 [MLLT10] and mixed-lineage leukemia-4 [MLLT4]) and two infant acute lymphocytic leukemia (ALL) samples were sensitive to menin inhibition. We also evaluated serial samples from two patients with multiply relapsed ALL. We found that highly pretreated KMT2A::AFF1 ALL samples were much less sensitive compared with cells obtained earlier in the same patients' disease course. Because none of the patients had been treated with a menin inhibitor, resistance in these highly pretreated samples was acquired in the absence of menin-inhibitor exposure. Transcriptomic analysis documented sustained on-target efficacy toward the canonical targets of the menin inhibitor in resistant cells. Targeted genomic analysis documented the emergence of multiple comutations, including RAS pathway and TP53 mutations, although neither was sufficient to induce menin-inhibitor resistance in vitro. Downregulation of KMT3D may account for resistance in one patient; inactivation of KMT2C/D has been reported to result in menin-inhibitor resistance, and KMT2C-edited cells from this patient were selected for in menin-inhibitor-containing growth conditions. Future studies will need to clarify more broadly which genomic/epigenomic alterations drive upfront resistance. Regardless of mechanism, our data support using menin inhibitors upfront or in early lines of therapy before substantial genomic or epigenomic evolution has occurred.