Supplemental Table 1. List of DNA gene mutations tested in diagnostic tumor samples of our ALL cohort. Supplemental Table 2. List of 80 genes with RNA sequencing coverage. Supplemental Table 3. Demographics of 400 adult ALL cases. Supplemental Table 4. List of first-line (initial) therapies in Ph-positive B-ALL, Ph-negative B-ALL, and Tlineage ALL patients. Supplemental Table 5. Baseline gene rearrangements and structural variants of 329 molecularly characterized adult B-ALL patients. Supplemental Table 6. Baseline gene rearrangements and structural variants of 71 molecularly characterized adult T-lineage ALL patients. Supplemental Table 7. List of pathogenic/likely pathogenic germline variants in our ALL cohort. Supplementary Table 8. Multivariable analysis of overall survival (OS) in B-ALL patients. Supplemental Table 9. Clinical characteristics of patients studied in scDNA + protein sequencing experiments. Supplemental Table 10. Clinical characteristics of patients studied in scRNA-seq experiment. Supplemental Table 11. Ingenuity pathway analysis comparing B-lymphoblasts from B-ALL with MyM vs B-ALL without MyM.

Supplemental Figure 1. WHO/ICC subtypes for the B-ALL cohort. Supplemental Figure 2. Lymphoid clonal hematopoiesis (CH) mutations are less common in adults with ALL. Supplemental Figure 3. Blast percentage in the pre-treatment sample and detected variant allelic frequencies (VAF) of mutations in ALL patients with myeloid mutations. Supplemental Figure 4. Germline testing in the adult ALL cohort. Supplemental Figure 5. Associations of gene mutations and WHO/ICC disease subtypes in B-ALL. Supplemental Figure 6. Overall survival in adults with B-ALL. Supplemental Figure 7. Kaplan-Meier overall survival curves for Ph-negative B-ALL patients stratified based on the presence of TP53 and non-TP53 MyM (A), and treatment with different chemotherapy protocols (B, C, D). LH, low hypodiploidy. Supplemental Figure 8. T-lineage ALL with myeloid mutations (MyM). Supplemental Figure 9. Expression levels of lineage-specific surface markers. Supplemental Figure 10. Single cell DNA and protein sequencing to study the clonal architecture of Tlineage ALL with MyM. Supplemental Figure 11. A, Flow plots showing ETP-lymphoblast, myeloid, and mature lymphocytic compartments of ETP-ALL1, which were sorted for DNA extraction and sequencing. B, Distribution of variant allelic frequencies (VAFs) for myeloid (IDH2, DNMT3A) and NRAS mutations across subpopulations of cells in ETP-ALL1. Supplemental Figure 12. Clonal dynamics of ALL with MyM. Supplemental Figure 13. Two CH mutations (TP53 and DNMT3A) were detectable seven years before the diagnosis of therapy-related ALL22. Supplemental Figure 14. Clonal evolution of B-ALL2. Supplemental Figure 15. Lymphoblasts from B-ALL with MyM are characterized by their resistance to cytotoxic chemotherapy. Supplemental Figure 16. Curves showing viability of primary human B-ALL samples with TP53 mutation (n= 14), MyM (n= 16) and no MyM/TP53 (n= 24), treated with vincristine, doxorubicin, and blinatumomab at escalating doses. Supplemental Figure 17. Flow cytometry plots showing ALL22 (TP53-mutated B-ALL) sample treated with escalating doses of blinatumomab. Supplemental Figure 18. A, CR with MRD negativity rates in Ph-negative B-ALL patients treated with pediatric regimens, stratified based on age, gender, and treatment site. B, CR with MRD negativity rates in Ph-negative B-ALL patients treated with hyper-CVAD, stratified based on age, gender, and treatment site. C, CR with MRD negativity rates in Ph-negative B-ALL patients treated with inotuzumab. Supplemental Figure 19. A, MHC class I and II antigen expression in blasts from B-ALL with MyM vs B-ALL without MyM/TP53. B, Comparison of genes implicated in blinatumomab resistance in B-ALL with vs without MyM/TP53.

Abstract Myeloid neoplasms arise from preexisting clonal hematopoiesis (CH); however, the role of CH in the pathogenesis of acute lymphoblastic leukemia (ALL) is unknown. We found that 18% of adult ALL cases harbored TP53, and 16% had myeloid CH-associated gene mutations. ALL with myeloid mutations (MyM) had distinct genetic and clinical characteristics, associated with inferior survival. By using single-cell proteogenomic analysis, we demonstrated that myeloid mutations were present years before the diagnosis of ALL, and a subset of these clones expanded over time to manifest as dominant clones in ALL. Single-cell RNA sequencing revealed upregulation of genes associated with cell survival and resistance to apoptosis in B-ALL with MyM, which responds better to newer immunotherapeutic approaches. These findings define ALL with MyM as a high-risk disease that can arise from antecedent CH and offer new mechanistic insights to develop better therapeutic and preventative strategies. Significance: CH is a precursor lesion for lymphoblastic leukemogenesis. ALL with MyM has distinct genetic and clinical characteristics, associated with adverse survival outcomes after chemotherapy. CH can precede ALL years before diagnosis, and ALL with MyM is enriched with activated T cells that respond to immunotherapies such as blinatumomab. See related commentary by Iacobucci, p. 142.

AbstractPurpose: Relapsed T-acute lymphoblastic leukemia (T-ALL) has limited treatment options. We investigated mechanisms of resistance to BH3 mimetics in T-ALL to develop rational combination strategies. We also looked at the preclinical efficacy of NWP-0476, a novel BCL-2/BCL-xL inhibitor, as single agent and combination therapy in T-ALL. Experimental Design: We used BH3 profiling as a predictive tool for BH3 mimetic response in T-ALL. Using isogenic control, venetoclax-resistant (ven-R) and NWP-0476-resistant (NWP-R) cells, phosphokinase array was performed to identify differentially regulated signaling pathways. Results: Typical T-ALL cells had increased dependence on BCL-xL, whereas early T-precursor (ETP)-ALL cells had higher BCL-2 dependence for survival. BCL-2/BCL-xL dual inhibitors were effective against both subtypes of T-lineage ALL. A 71-protein human phosphokinase array showed increased LCK activity in ven-R cells, and increased ACK1 activity in ven-R and NWP-R cells. We hypothesized that pre-TCR and ACK1 signaling pathways are drivers of resistance to BCL-2 and BCL-xL inhibition, respectively. First, we silenced LCK gene in T-ALL cell lines, which resulted in increased sensitivity to BCL-2 inhibition. Mechanistically, LCK activated NF-κB pathway and the expression of BCL-xL. Silencing ACK1 gene resulted in increased sensitivity to both BCL-2 and BCL-xL inhibitors. ACK1 signaling upregulated AKT pathway, which inhibited the pro-apoptotic function of BAD. In a T-ALL patient-derived xenograft model, combination of NWP-0476 and dasatinib demonstrated synergy without major organ toxicity. Conclusions: LCK and ACK1 signaling pathways are critical regulators of BH3 mimetic resistance in T-ALL. Combination of BH3 mimetics with tyrosine kinase inhibitors might be effective against relapsed T-ALL.