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Although acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, it is unknown how or which host immune factors influence the long-term remission of this cancer. To this end, we systematically evaluated the effects of T-cell immunity on Ph+ ALL therapy outcomes. Using a murine Arf−/−BCR-ABL1 B-cell ALL model, we showed that loss of T cells in the host drastically increased leukemia relapse after dasatinib or cytotoxic chemotherapy. Although ABL1 mutations emerged early during dasatinib treatment in both immunocompetent and immunocompromised hosts, T-cell immunity was essential for suppressing the outgrowth of drug-resistant leukemia. Bulk and single-cell transcriptome profiling of T cells during therapy pointed to the activation of type 1 immunity-related cytokine signaling being linked to long-term leukemia remission in mice. Consistent with these observations, interferon γ and interleukin 12 directly modulated dasatinib antileukemia efficacy in vivo. Finally, we evaluated peripheral blood immune cell composition in 102 children with ALL during chemotherapy and observed a significant association of T-cell abundance with treatment outcomes. Together, these results suggest that T-cell immunity plays pivotal roles in maintaining long-term remission of ALL, highlighting that the interplay between host immunity and drug resistance can be harnessed to improve ALL chemotherapy outcomes.

Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor–mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a–mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.

While most childhood acute lymphoblastic leukemia (ALL) can be cured with risk-adapted combination chemotherapy, a significant proportion of patients experience relapse and eventually succumb to leukemia. In fact, ~50% of ALL relapses occur in patients who have excellent early treatment response (i.e., negative minimal residual disease [MRD] at the end of induction therapy), suggesting that long-term remission is determined by factors beyond leukemia cell drug sensitivity. Inherited genetic variants in genes related to host immune functions have been linked to ALL prognosis, pointing to a possible role of immune vigilance against residual leukemia. However, little is known about how and which host immune factors influence the long-term-remission of this cancer. To this end, we first compared leukemia-free survival of murine BCR-ABL1 B-ALL in response to chemotherapy, in immunocompetent mice (C57BL/6J, "B6") or mice with various T cell deficiencies. Complete loss of all T cells in the host (Tcra KO) led to a drastic increase in leukemia relapse after treatment with ABL inhibitor dasatinib (p=0.0014) or cytotoxic chemotherapy (e.g., dexamethasone [p=0.0002], and mercaptopurine [p=0.0106]). Similar results were observed when we specifically depleted CD4 or CD8 T cells in host mice (p=0.0302 and 0.0010 for CD4 and CD8 depletion, respectively). ABL1 mutation analysis showed that the immunocompetent mice were much more likely to relapse with wild-type BCR-ABL1 B-ALL after dasatinib withdrawal (71.4%). By contrast, most immunocompromised mice (60.0%, 75%, and 80% of TcraKO, CD4 T cell depletion, and CD8 T cell depletion) relapsed with ABL1-T315I B-ALL. Deep sequencing of BCR-ABL1 mutations during dasatinib therapy indicated that drug resistance mutations emerged early in leukemia regardless of immunocompetency of the host, but the lack of T cell immunity strongly predisposed mice to subsequently develop drug resistance and thus relapse. Furthermore, immunocompetent mice that achieved long-term remission also rejected re-engraftment of BCR-ABL1 ALL, suggesting immune memory against leukemia established during dasatinib treatment. To explore the molecular basis of T-cell immunity against ALL, we collected peripheral T cells during dasatinib therapy from mice which were eventually cured vs. those experienced relapse and compared their global gene expression pattern. Through expression array-based profiling of bulk T cells, we noted upregulation of memory/effector T cell signature genes in mice achieving long-term cure (especially in CD8 T cells), along with genes associated with TCR signaling and cytokine response (IL12 and IFNG). Similarly, scRNA-seq profiling also confirmed the association of TCR signaling, IL12 and IFNG signaling activation in T cells with the cure of ALL in mice. Single-cell RNA-seq analyses of T cell subpopulation specifically identified differences in memory-like T cells from relapse mice vs. those cured with the same chemotherapy, with the latter showing more robust activation of TCR and cytokine signalings. We thus hypothesized that IL12 and IFNG response signaling are key determinants of successful immune surveillance against ALL. Indeed, our in vivo experiments confirmed that 1) IfngKO mice relapsed much more quickly during dasatinib treatment with drug resistance BCR-ABL1 mutations than immunocompetent mice (p Finally, we validated these findings by evaluating the immune cell composition of peripheral blood samples collected during remission from 105 children with ALL treated on the St. Jude Total Therapy Study XV clinical trial. Using the gene expression-based deconvolution method, we quantified the frequency of 9 types of immune cells. There was a significant association of higher level of total T cell (CD8, CD4 naïve, CD4 memory, follicular helper, regulatory, and gamma delta T cells) with superior event-free survival (p=0.031), whereas other immune cells had no impact on ALL treatment outcomes. Even after adjusting for ALL molecular subtypes and MRD levels, T cell frequency remained prognostic (p=0.034). In conclusion, our results firmly established the role of T cell immunity in the cure of ALL and also pointed to T cell activation as a means of improving treatment outcomes of ALL. Disclosures Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee.

Introduction: Activating internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) is one of the most frequently mutations in acute myeloid leukemia (AML) patients with a frequency of 20-30%. Although AML with FLT3-ITD mutation is not recognized as a distinct entity in the World Health Organization classification system, AML patients with FLT3-ITD usually have high white blood cell count, high risk in poor prognostic outcome and are benefited from FLT3 inhibitors. However, the outcome prediction of AML with FLT3-ITD is still not very clear in patients undergoing allogeneic hematopoietic cell transplantation (alloHCT). Recent studies showed that the depth of remission, mostly accessed by flow cytometry (FCM) based measurable residual disease (MRD) and next generation sequencing based molecular MRD, was highly predictive for relapse in AML before alloHCT. The depth of remission is considered an important criterion for evaluating AML relapse before alloHCT. Hence, FLT3-ITD remission depth showed a potential application in evaluating FLT3-ITD subtype's prognosis. Therefore, in this retrospective study we tried to evaluate prognostic impact of FLT3-ITD remission depth in AML before alloHCT. Methods: A total of 117 FLT3-ITD positive patients were obtained in our study if they were 18 years or older, had a diagnosis of AML excluding acute promyelocytic leukemia, received standard induction chemotherapy comprising anthracycline and cytoarabine, at first affiliated hospital of Soochow University between September 2011 to January 2018 . All patients were examined a panel of 51 genes mutation by next generation sequencing and FLT3-ITD allelic ratio (AR) in newly diagnosed bone marrow samples (Fig1 A). 57 patients received two courses of standard chemotherapy and immediate alloHSCT in four months. The median time from first chemotherapy to alloHSCT was 99 days. FLT3-ITD was also examined direct after secondary chemotherapy with the sensitivity level of 0.005. Distribution of OS and RFS were estimated by Kaplan-Meier method. Difference of FLT3-ITD positive ratio in FLT3-ITD subtypes was analyzed by Chi-squared test. The two-sided level of significance was set at p Results: In our cohort, the median FLT3-ITD AR of 117 patients was 0.53 (range 0.02-7.72) in newly diagnosis. Interestingly, patients with DNMT3A mutation (0.615, range 0.04-7.72) had a higher FLT3-ITD AR than those of patients with CEBPA double mutation (0.19, range 0.02-0.74), with statistical difference (p=0.007). Patients with DNMT3A mutation also had lower complete remission rate than those of patients with NPM1 mutation and DNMT3A wildtype after first chemotherapy (x2=5.987, P=0.018). In 57 patients undergoing alloHSCT, we found there were no differences in OS and RFS among patients classified by FLT3-ITD AR or concomitant NPM1 mutation. However, patients with FLT3-ITD positive had a poorer OS (p0.01 had a poorer OS and EFS than those of patients with FLT3-ITD AR In conclusion, our results suggested FLT3-ITD remission depth is useful in prognosis prediction for AML patients with FLT3-ITD before alloHCT. Figure Disclosures No relevant conflicts of interest to declare.