The human hemopoietic progenitor hierarchy producing lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but remain incompletely characterized. Here, we demonstrated cord blood lympho-myeloid containing progenitor populations - the lymphoid-primed multi-potential progenitor (LMPP), granulocyte-macrophage progenitor (GMP) and multi-lymphoid progenitor (MLP) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Though most progenitors had uni-lineage myeloid or lymphoid potential, bi- and rarer multi-lineage progenitors occurred in LMPP, GMP and MLP. This, coupled with single cell expression analyses, suggested a continuum of progenitors execute lymphoid and myeloid differentiation rather than only uni-lineage progenitors being present downstream of stem cells.

Highlights d Genetic and functional analyses of myeloid preleukemia and leukemia in Down syndrome d Non-GATA1 preleukemic mutations are often not required for preleukemia d Previously undescribed transforming hotspot mutation in CSF2RB identified d Loss of function of 18 genes validated in transformation of preleukemia to leukemia

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.

Quek and colleagues identify human leukemic stem cells (LSCs) present in CD34− AML. In-depth characterization of the functional and clonal aspects of CD34− LSCs indicates that most are similar to myeloid precursors.

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Highlights d Biallelic C/EBPa and GATA-2 ZnF1 mutations synergize during leukemogenesis d GATA-2 ZnF1 mutation generates an erythroid-permissive chromatin state d C/EBPa and GATA-2 mutant NMPs show ectopic erythroid lineage potential d Transformed leukemic NMPs are bipotent neutrophilerythroid leukemia-initiating cells

7012 Background: Isocitrate dehydrogenase-1 ( IDH1+) mutations are present in 5-15% of myeloid malignancies, promoting leukemogenesis through production of the oncometabolite 2-hydroxyglutarate resulting in arrested myeloid differentiation. IDH1+ malignancies demonstrate increased reliance on the anti-apoptotic protein BCL-2, enhancing susceptibility to the BCL-2 inhibitor venetoclax (VEN). We report an interim safety and efficacy analysis of the IDH1 inhibitor ivosidenib (IVO; 500 mg PO daily D15-continuous) combined with VEN (D1-14) +/- azacitidine (AZA; 75mg/m2 D1-7 every 28 days). Methods: Eligible patients age ≥18 with IDH1+ MDS, newly diagnosed AML (ND: treatment naïve [TN] or secondary/treated secondary AML [sAML]), or relapsed/refractory (R/R) AML enrolled into three dose levels (DL): DL1 (IVO+VEN 400 mg), DL2 (IVO+VEN 800 mg), DL3 (IVO+VEN 400 mg+AZA). Primary objectives included safety and tolerability, and IWG defined overall response (ORR: CR+CRi+CRh+PR+MLFS). Prior receipt of IVO or VEN was exclusionary. Results: 25 evaluable patients (DL1: 6, DL2: 6, DL3: 13) enrolled with a median follow-up of 16.1 months. Median age was 67 (range: 44-84). 84% (N=21) of patients had AML (ND: N=13 [TN: 8, sAML: 5], R/R: N=8), while 16% (N=4) had MDS. ELN risk was intermediate and adverse in 16% (N=4) and 56% (N=14). Median IDH1 VAF at enrollment was 22.7% (range: 5.1%-47.8%). Two patients had received a prior IDH1 inhibitor. The ORR was 92% (DL1: 67%, DL2: 100%, DL3: 100%). Composite CR (CRc: CR+CRi+CRh) was 84% (DL1: 67%, DL2: 100%, DL3: 85%) including 92% (TN: 100%, sAML: 80%), 63%, and 100% of patients with ND-AML, R/R-AML, or MDS. Median number of cycles received was 4 (DL1: 8.5, DL2: 6, DL3: 4) with ongoing responses in 62% (DL1: 33%, DL2: 50%, DL3: 82%) at 1-year. 8 patients transitioned to SCT (DL1: 0, DL2: 2, DL3: 6), and 8 patients remain on study (DL1: 2, DL2: 1, DL3: 5). 1-year OS was 68% for the entire study population (DL1: 50%, DL2: 67%, DL3: 78%), 71% in ND-AML (TN: 86%, sAML: 60%), 50% in R/R-AML, and 100% in MDS. Measurable residual disease negative CRc by multiparameter flow cytometry was attained in 60% (ND-AML: 67%, R/R-AML: 60%, MDS: 33%) correlating with improved OS (median OS: NR vs. 8.5 months, p-value: 0.038). Common grade 3/4 adverse events included febrile neutropenia (28%) and pneumonia (24%). Tumor lysis and differentiation syndrome occurred in two and four patients; all cases resolved with medical management. Conclusions: IVO+VEN +/- AZA is an effective treatment regimen in patients with IDH1+myeloid malignancies. The combination therapy is associated with an acceptable and expected toxicity profile with notable efficacy and high rates of MRD-negative CRc in AML. Enrollment into the study continues. Clinical trial information: NCT03471260. [Table: see text]

7500 Background: Mutations in the isocitrate dehydrogenase-1 gene ( IDH1) result in myeloid differentiation arrest and accumulation of the oncometabolite 2-hydroxyglutarate (2-HG), promoting leukemogenesis. We report a primary safety and efficacy analysis of the IDH1 inhibitor ivosidenib (IVO; 500 mg PO daily D15-continous) combined with venetoclax (VEN; D1-14 per 28-day cycle), with and without azacitidine (AZA; 75mg/m2 D1-7). Methods: Eligible patients age ≥18 with IDH1 mutated myeloid malignancies (high-risk MDS and AML) enrolled into one of three successive cohorts (Cohort 1: IVO+VEN 400 mg, Cohort 2: IVO+VEN 800 mg, Cohort 3: IVO+VEN 400 mg+AZA). Primary endpoints include safety and tolerability and overall response rate (ORR) by revised IWG criteria. Key secondary endpoints include survival endpoints and PK correlates. Results: 19 patients (median age 68) enrolled, 17 with AML: 9 relapsed/refractory AML (R/R; median 1 prior line of therapy), 5 treatment naïve AML, and 3 HMA-failure MDS with secondary AML. Two patients had high-risk MDS. ELN risk was favorable, intermediate, and adverse risk in 37%, 15%, and 47%. Co-mutations included NPM1 (37%), chromatin-spliceosome (32%), methylation (16%), and RAS pathway (21%). Adverse events of special interest included IDH differentiation syndrome (n=4, grade > 3 in 1) and tumor lysis syndrome (TLS; n=2), including one grade 3 TLS event in a NPM1+ patient (successfully managed without hemodialysis). In evaluable patients (n=18), composite complete remission (CRc: CR+CRi+CRh) rates were 78% overall (treatment naive: 100%, R/R: 75%), and 67%, 100%, and 67% by cohort (median time to best response: 2 months). 7 (50%) patients achieving CRc were also MRD negative by flow cytometry. 1 patient had HI without CR/CRi and 1 had a MLFS. 9 (50%) patients remain on study, 3 (17%) proceeded to SCT in CR, 2 were non-responders, and 5 (22%) experienced progressive disease following CRc occurring after a median of 3 months. After a median follow up of 3.5 months, median OS was not reached in treatment naïve patients, and 9.7 months in R/R patients. Conclusions: IVO+VEN +AZA therapy is well tolerated and highly effective for patients with IDH1 mutated AML. Follow up and accrual is ongoing to better define duration and biomarkers of response. Clinical trial information: NCT03471260 . [Table: see text]