MLL gene rearrangements are well-recognized aberrations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In contrast, MLL gene amplification in AML/MDS remains poorly characterized. Here, we report a series of 21 patients with myeloid neoplasms associated with MLL gene amplification from 1 institution. This series included 13 men and 8 women, with a median age of 64 years. Eleven patients presented as AML with myelodysplasia-related changes, 6 as therapy-related AML, and 4 as therapy-related MDS. All patients had a highly complex karyotype, including frequent -5/del(5q), -18, and -17/del(17p) abnormalities; 16 patients were hypodiploid. TP53 mutations were detected in all 12 patients tested, and 3 patients showed TP53 mutation before MLL amplification. Morphologically, the leukemic cells frequently showed cytoplasmic vacuoles, bilobed nuclei, and were associated with background dyspoiesis. Immunophenotypically, 15 patients had a myeloid and 4 had myelomonocytic immunophenotype. Laboratory coagulopathies were common; 7 patients developed disseminated intravascular coagulopathy, and 3 died of intracranial bleeding. All patients were refractory to therapy; the median overall survival was 1 month, after MLL gene amplification was detected. We concluded that AML/MDS with MLL gene amplification is likely a subset of therapy-related AML/MDS or AML with myelodysplasia-related changes, associated with distinct clinicopathological features, frequent disseminated intravascular coagulopathy, a highly complex karyotype, TP53 deletion/mutation, and an aggressive clinical course.

Nearly one-third of patients with acute myeloid leukemia have FMS-like tyrosine kinase 3 mutations and thus have poor survival prospects. Receptor tyrosine kinase anexelekto is critical for FMS-like tyrosine kinase 3 signaling and participates in FMS-like tyrosine kinase 3 inhibitor resistance mechanisms. Thus, strategies targeting anexelekto could prove useful for acute myeloid leukemia therapy. ONO-7475 is an inhibitor with high specificity for anexelekto and MER tyrosine kinase. Herein, we report that ONO-7475 potently arrested growth and induced apoptosis in acute myeloid leukemia with internal tandem duplication mutation of FMS-like tyrosine kinase 3. MER tyrosine kinase-lacking MOLM13 cells were sensitive to ONO-7475, while MER tyrosine kinase expressing OCI-AML3 cells were resistant, suggesting that the drug acts via anexelekto in acute myeloid leukemia cells. Reverse phase protein analysis of ONO-7475 treated cells revealed that cell cycle regulators like cyclin dependent kinase 1, cyclin B1, polo-like kinase 1, and retinoblastoma were suppressed. ONO-7475 suppressed cyclin dependent kinase 1, cyclin B1, polo-like kinase 1 gene expression suggesting that anexelekto may regulate the cell cycle, at least in part, via transcriptional mechanisms. Importantly, ONO-7475 was effective in a human FMS-like tyrosine kinase 3 with internal tandem duplication mutant murine xenograft model. Mice fed a diet containing ONO-7475 exhibited significantly longer survival and, interestingly, blocked leukemia cell infiltration in the liver. In summary, ONO-7475 effectively kills acute myeloid leukemia cells in vitro and in vivo by mechanisms that involve disruption of diverse survival and proliferation pathways.

T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, n = 44) and extranodal NK/T-cell lymphomas (ENKTLs, n = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination.

Incubation of umbilical cord blood (UCB) derived regulatory T-cells (Tregs) with fucosyltransferase enzyme improves their ability to home to the target tissue to prevent graft vs. host disease (GVHD). We report results of 5 patients (Double UCB Transplant, n=2; Peripheral Blood Matched Unrelated Donor Transplant, n=3) who received UCB-Tregs (Dose level = 1×106/kg), infused one day prior to the donor graft. All patients received their designated UCB-Treg dose without any infusion reaction. The ratio of conventional T-cells in donor graft was at least 10 times higher than infused UCB-Tregs (ratio range, 12-356). All patients engrafted at median of 13 days (range, 8-17 days). One patient died due to brain hemorrhage on day 45. A bi-modal increase of plasma IL-10 level occurred on day 7 and day 21 and notably, plasma IL-2 level dropped significantly in all patients at Day 7. All evaluable patients developed ≥grade II acute GVHD and at 1 year follow up, all were alive and without evidence of disease relapse. No increase in the chronic GVHD biomarkers (REG3a and Elafin) was observed at day 7. At the time of last follow up, all evaluable patients were off immune-suppression. Stage 2 of this clinical trial examining UCB-Treg at dose level= 1×107/kg is currently underway.

Fluorescence in situ hybridization (FISH) is a confirmatory test to establish a diagnosis of inv(16)/t(16;16) AML. However, incidental findings and their clinical diagnostic implication have not been systemically studied. We studied 1629 CBFB FISH cases performed in our institution, 262 (16.1%), 1234 (75.7%), and 133 (8.2%) were reported as positive, normal, and abnormal, respectively. The last included CBFB copy number changes (n = 120) and atypical findings such as 3'CBFB deletion (n = 11), 5'CBFB deletion (n = 1), and 5'CBFB gain (n = 1). Correlating with CBFB-MYH11 RT-PCR results, totally 271 CBFB rearrangement cases were identified, including five with discrepancies between FISH and RT-PCR due to new partner genes (n = 3), insertion (n = 1), or rare CBFB-MYH11 variant (n = 1) and eight with 3'CBFB deletion. All cases with atypical findings and/or discrepancies presented clinical diagnostic challenges. Correlating FISH signal patterns and karyotypes, additional chromosome 16 aberrations (AC16As) show impacts on the re-definition of a complex karyotype and prognostic prediction. The CBFB rearrangement but not all AC16As will be detected by NGS-based methods. Therefore, FISH testing is currently still needed to provide a quick and straightforward confirmatory inv(16)/t(16;16) AML diagnosis and additional information related to clinical management.

Patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN) have poor outcomes despite intensive chemotherapy, underscoring the need for novel therapeutic approaches. The expression status of PD1/PD-L1 in BPDCN remains unknown. We evaluated PD1/PD-L1 by immunohistochemistry and RNAseq expression profiling in a cohort of BPDCN patients. The study group included 28 patients with a median age of 66.8 years (range, 22.8-86.7), 22 men and 6 women. PD-L1 expression was detected by immunohistochemistry in 10/21 (47.6%) cases. PD-L1 expression had a median H-score of 157. The H-score was ≥60 in 7 patients. PD-L1 protein levels (H-score) were proportional to normalized PD-L1 mRNA transcript levels (CD274 mRNA). In addition, high-level PD-L1 expression correlated with higher numbers of PD1-positive cells within BPDCN tumors. There was no correlation between clinicopathologic characteristics and PD-L1 expression status. Similarly, there was no significant difference in overall survival between patients with PD-L1-positive and PD-L1-negative BPDCN (median 12 vs. 23 month, respectively; p = 0.743). In conclusion, PD-L1 expression by tumor cells is detectable in a sizeable subset of patients with BPDCN, suggesting that exploration of the effectiveness of therapeutic inhibition of the PD1/PD-L1 axis in patients with refractory or progressive BPDCN is warranted.

MYD88 mutations in chronic lymphocytic leukemia (CLL) are not well characterized. Earlier reports yielded conflicting results in terms of clinicopathologic presentation and prognostic impact of MYD88 mutations in CLL patients. In addition, the morphological and immunophenotypic features of CLL cases carrying MYD88 mutations have not been explored. Finally, the clinical or biologic implications of the canonical L265P MYD88 mutation vs. mutations in other sites of MYD88 within the context of CLL are also unknown. In this study, a cohort of 1779 CLL patients underwent mutational analysis, and 56 (3.1%) cases were found to have MYD88 mutations, including 38 with L265P mutations (designated here as group A) and 18 with non-L265P mutations (group B). Cases with wild type MYD88 were included as controls. There was no morphological difference in cases with and without MYD88 mutations. Immunophenotypically, cases with mutated MYD88 (both groups A and B) more frequently had an atypical immunophenotype when compared to wild type cases. Group A patients were younger and were associated with variable favorable prognostic factors, including less elevated β2-microglobulin level, negative CD38 and ZAP70, higher frequency of mutated IGHV and isolated del(13q14.3), and lower frequency of del(11q22.3) and mutations of NOTCH1 and SF3B1. In contrast, group B patients were more similar to CLL patients with wild type MYD88. There was no difference in time to first treatment when comparing MYD88-mutated vs. wild type CLL patients before and after stratification according to IGHV mutation status. In summary, MYD88 mutations are uncommon in CLL and cases with L265P mutation have distinctive clinical, immunophenotypic, cytogenetic, and molecular features. There is no significant impact of MYD88 mutations on time to first treatment in CLL.

FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in acute myeloid leukemia (AML). The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor, ONO-7475, kills FLT3-mutant AML cells with targets including Extracellular- signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single in vivo agent using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO-7475/ABT-199 was even more potent in reducing leukemic burden and prolonging the survival of mice in both model systems. These results suggest that the ONO-7475/ABT-199 combination may be effective for AML therapy.

Plasmablastic lymphoma mutational profile is undescribed. Here we performed a targeted exonic NGS analysis of 30 plasmablastic lymphoma cases with a B cell lymphoma dedicated panel and FISH for the detection of MYC rearrangements. A complete phenotyping of the neoplastic and microenvironment cell populations was also performed. We have identified an enrichment in recurrent genetic events in MYC (69% with MYC translocation or amplification and 3 cases with missense point mutations), PRDM1/Blimp1 and STAT3 mutations. These gene mutations were more frequent in EBV positive disease. Other genetic events included mutations in BRAF, EP300, BCR (CD79A and CD79B), NOTCH pathway (NOTCH2, NOTCH1 and SGK1) and MYD88pL265P. Immunohistochemical analysis showed consistent MYC expression, higher in cases with MYC rearrangements together with phospho-STAT3 (Tyr705) overexpression in cases with STAT3 SH2 domain mutations. Microenvironment populations were heterogeneous and unrelated with EBV, with an enrichment of Tumor Associated Macrophages (TAM) and PD1 positive T cells. PD-L1 was expressed in all cases in the TAM population but only in 5 cases in the neoplastic cells (4 out of 14 EBV positive cases). HLA expression was absent in the majority of PBL cases. In summary, Plasmablastic lymphoma mutational profile is heterogeneous and related with EBV infection. Genetic events in MYC, STAT3 and PRDM1/Blimp1 are more frequent in EBV positive disease. An enrichment in TAM and PD1 reactive T lymphocytes is found in the microenvironment of PBL cases, that express PD-L1 in the neoplastic cells in a fraction of cases.

Richter Transformation (RT) develops in CLL as an aggressive, therapy-resistant, diffuse large B cell lymphoma (RT-DLBCL), commonly clonally-related (CLR) to the concomitant CLL. Lack of available pre-clinical human models has hampered the development of novel therapies for RT-DLBCL. Here, we report the profiles of genetic alterations, chromatin accessibility and active enhancers, gene-expressions and anti-lymphoma drug-sensitivity of three newly established, patient-derived, xenograft (PDX) models of RT-DLBCLs, including CLR and clonally-unrelated (CLUR) to concomitant CLL. The CLR and CLUR RT-DLBCL cells display active enhancers, higher single-cell RNA-Seq-determined mRNA, and protein expressions of IRF4, TCF4, and BCL2, as well as increased sensitivity to BET protein inhibitors. CRISPR knockout of IRF4 attenuated c-Myc levels and increased sensitivity to a BET protein inhibitor. Co-treatment with BET inhibitor or BET-PROTAC and ibrutinib or venetoclax exerted synergistic in vitro lethality in the RT-DLBCL cells. Finally, as compared to each agent alone, combination therapy with BET-PROTAC and venetoclax significantly reduced lymphoma burden and improved survival of immune-depleted mice engrafted with CLR-RT-DLBCL. These findings highlight a novel, potentially effective therapy for RT-DLBCL.

Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a surrogate source of tumor material that can be obtained via a venipuncture (also known as a liquid biopsy) and used to better understand tumor characteristics. However, the only FDA-cleared CTC assay has been limited to the enumeration of surface marker-defined cells and not further characterization of the CTCs. In this study, we tested the ability of a semi-automated device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, agnostic of cell-surface markers (the Parsortix® PC1 System), to yield CTCs for evaluation by downstream techniques commonly available in clinical laboratories. The data generated from this study were used to support a De Novo request (DEN200062) for the classification of this device, which the FDA recently granted. As part of a multicenter clinical trial, peripheral blood samples from 216 patients with metastatic breast cancer (MBC) and 205 healthy volunteers were subjected to CTC enrichment. A board-certified pathologist enumerated the CTCs from each participant by cytologic evaluation of Wright-Giemsa-stained slides. As proof of principle, cells harvested from a concurrent parallel sample provided by each participant were evaluated using one of three additional evaluation techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. The study demonstrated that the Parsortix® PC1 System can effectively capture and harvest CTCs from the peripheral blood of MBC patients and that the harvested cells can be evaluated using orthogonal methodologies such as gene expression and/or Fluorescence In Situ Hybridization (FISH).

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a clinically aggressive blood cancer, often involving the skin, bone marrow, lymph nodes, and central nervous system (CNS) in 20% to 30% of patients. Despite significant progress in CD123- and BCL-2-targeted therapy, most patients are not cured without hematopoietic stem cell transplant (HSCT), and CNS relapses occur quite frequently. Combination approaches with targeted and chemotherapy agents plus incorporation of prophylactic CNS-directed therapy are urgently needed. In this setting, we sought to analyze outcomes using the cytotoxic chemotherapy backbone regimen hyperfractionated cyclophosphamide, vincristine, adriamycin, and dexamethasone (HCVAD). We conducted a retrospective analysis of patients with BPDCN (n = 100), evaluating complete remission (CR) and median overall survival (OS) among 3 groups: those who received frontline HCVAD-based therapy (n = 35), SL-401 (n = 37), or other regimens (n = 28). HCVAD-based regimens yielded higher CR (80% vs 59% vs 43%; P = .01). There was no significant difference in OS (28.3 vs 13.7 vs 22.8 months; P = .41) or remission duration probability among treatment groups (38.6 vs not reached vs 10.2 months; P = .24). HSCT was performed in 51% vs 49% vs 38%, respectively (P = .455). These results suggest a continued important role for HCVAD-based chemotherapy in BPDCN, even in the modern targeted-therapy era, with high CR rates in the frontline setting. Further studies must establish the clinical activity, feasibility, and safety of doublet/triplet combinations of targeted therapies plus cytotoxic agents and the addition of CNS prophylaxis, with the ultimate goal of durable long-term remission for patients with BPDCN.

We present CellSpatialGraph, an integrated clustering and graph-based framework, to investigate the cellular spatial structure. Due to the lack of a clear understanding of the cell subtypes in the tumor microenvironment, unsupervised learning is applied to uncover cell phenotypes. Then, we build local cell graphs, referred to as supercells, to model the cell-to-cell relationships at a local scale. After that, we apply clustering again to identify the subtypes of supercells. In the end, we build a global graph to summarize supercell-to-supercell interactions, from which we extract features to classify different disease subtypes.

Cancers often overexpress multiple clinically relevant oncogenes, but it is not known if combinations of oncogenes in cellular subpopulations within a cancer influence clinical outcomes. Using quantitative multispectral imaging of the prognostically relevant oncogenes MYC, BCL2, and BCL6 in diffuse large B-cell lymphoma (DLBCL), we show that the percentage of cells with a unique combination MYC+BCL2+BCL6- (M+2+6-) consistently predicts survival across four independent cohorts (n = 449), an effect not observed with other combinations including M+2+6+. We show that the M+2+6- percentage can be mathematically derived from quantitative measurements of the individual oncogenes and correlates with survival in IHC (n = 316) and gene expression (n = 2,521) datasets. Comparative bulk/single-cell transcriptomic analyses of DLBCL samples and MYC/BCL2/BCL6-transformed primary B cells identify molecular features, including cyclin D2 and PI3K/AKT as candidate regulators of M+2+6- unfavorable biology. Similar analyses evaluating oncogenic combinations at single-cell resolution in other cancers may facilitate an understanding of cancer evolution and therapy resistance.

RAF molecules play a critical role in cell signaling through their integral impact on the RAS/RAF/MEK/ERK signaling pathway, which is constitutively activated in a sizeable subset of acute myeloid leukemia (AML) patients. We evaluated the impact of pan-RAF inhibition using LY3009120 in AML cells harboring mutations upstream and downstream of RAF. LY3009120 had anti-proliferative and pro-apoptotic effects and suppressed pERK1/2 levels in leukemic cells with RAS and FLT3 mutations. Using reverse protein phase array analysis, we identified reductions in the expression/activation of cell signaling components downstream of RAF (activated p38) and cell cycle regulators (Wee1/cyclin B1, Cdc2/Cdk1, activated Rb, etc.). Notably, LY3009120 potentiated the effect of Ara-C on AML cells and overcame bone marrow mesenchymal stromal cell-mediated chemoresistance, with RAS-mutated cells showing a notable reduction in pAKT (Ser473). Furthermore, the combination of LY3009120 and sorafenib resulted in significantly higher levels of apoptosis in AML cells with heterozygous and hemizygous FLT3 mutations. In conclusion, pan-RAF inhibition in AML using LY3009120 results in anti-leukemic activity, and combination with Ara-C or sorafenib potentiates its effect.

A 52-yr-old woman presented with therapy-related acute myeloid leukemia. A bone marrow biopsy showed 21% blasts with a myeloid phenotype and no other notable features such as abnormal eosinophils. Routine nanofluidics-based reverse transcriptase polymerase chain reaction (PCR) leukemia translocation panel designed to screen for recurrent genetic abnormalities in acute leukemia detected an inversion 16 transcript variant E. This prompted rereview of karyotype and fluorescence in situ hybridization studies, which confirmed inv(16), leading to appropriate prognostication and modification of treatment. This case underscores the utility of a powerful molecular screening method for the routine detection of recurrent genetic abnormalities of acute myeloid leukemia. It was especially useful in this case because of the lack of characteristic morphologic findings seen in inversion 16 and the difficulty in its detection by conventional karyotype analysis.

Germline, mono-allelic mutations in RUNX1 cause familial platelet disorder (RUNX1-FPD) that evolves into myeloid malignancy (FPD-MM): MDS or AML. FPD-MM commonly harbors co-mutations in the second RUNX1 allele and/or other epigenetic regulators. Here we utilized patient-derived (PD) FPD-MM cells and established the first FPD-MM AML cell line (GMR-AML1). GMR-AML1 cells exhibited active super-enhancers of MYB, MYC, BCL2 and CDK6, augmented expressions of c-Myc, c-Myb, EVI1 and PLK1 and surface markers of AML stem cells. In longitudinally studied bone marrow cells from a patient at FPD-MM vs RUNX1-FPD state, we confirmed increased chromatin accessibility and mRNA expressions of MYB, MECOM and BCL2 in FPD-MM cells. GMR-AML1 and PD FPD-MM cells were sensitive to homoharringtonine (HHT or omacetaxine) or mebendazole-induced lethality, associated with repression of c-Myc, EVI1, PLK1, CDK6 and MCL1. Co-treatment with MB and the PLK1 inhibitor volasertib exerted synergistic in vitro lethality in GMR-AML1 cells. In luciferase-expressing GMR-AML1 xenograft model, MB, omacetaxine or volasertib monotherapy, or co-treatment with MB and volasertib, significantly reduced AML burden and improved survival in the immune-depleted mice. These findings highlight the molecular features of FPD-MM progression and demonstrate HHT, MB and/or volasertib as effective agents against cellular models of FPD-MM.

First-generation bromodomain extra-terminal protein (BETP) inhibitors (BETi) (e.g., OTX015) that disrupt binding of BETP BRD4 to chromatin transcriptionally attenuate AML-relevant progrowth and prosurvival oncoproteins. BETi treatment induces apoptosis of AML BPCs, reduces in vivo AML burden and induces clinical remissions in a minority of AML patients. Clinical efficacy of more potent BETis, e.g., ABBV-075 (AbbVie, Inc.), is being evaluated. Venetoclax and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis. BETi treatment is shown here to perturb accessible chromatin and activity of enhancers/promoters, attenuating MYC, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A expressions and apoptosis of AML cells. Treatment with venetoclax increased MCL1 protein levels, but cotreatment with ABBV-075 reduced MCL1 and Bcl-xL levels. ABBV-075 cotreatment synergistically induced apoptosis with venetoclax or A-1210477 in patient-derived, CD34+ AML cells. Compared to treatment with either agent alone, cotreatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell-burden and improving survival, without inducing toxicity, in AML-engrafted immune-depleted mice. These findings highlight the basis of superior activity and support interrogation of clinical efficacy and safety of cotreatment with BETi and BCL2 or MCL1 inhibitor in AML.

Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory T cells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key T cell genes (TCF7, LMO2, CD69) and impact pathways of T cell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683high T cells with response.