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Three patients with progressive multifocal leukoencephalopathy who were treated with third-party–produced, cryopreserved, partially HLA-matched T cells specific for BK virus had clinical improvement or stabilization of PML and reduction in JC viral load.

Sample barcoding is essential in mass cytometry analysis, since it can eliminate potential procedural variations, enhance throughput, and allow simultaneous sample processing and acquisition. Sample pooling after prior surface staining termed live-cell barcoding is more desirable than intracellular barcoding, where samples are pooled after fixation and permeabilization, since it does not depend on fixation-sensitive antigenic epitopes. In live-cell barcoding, the general approach uses two tags per sample out of a pool of antibodies paired with five palladium (Pd) isotopes in order to preserve appreciable signal-to-noise ratios and achieve higher yields after sample deconvolution. The number of samples that can be pooled in an experiment using live-cell barcoding is limited, due to weak signal intensities associated with Pd isotopes and the relatively low number of available tags. Here, we describe a novel barcoding technique utilizing 10 different tags, seven cadmium (Cd) tags and three Pd tags, with superior signal intensities that do not impinge on lanthanide detection, which enables enhanced pooling of samples with multiple experimental conditions and markedly enhances sample throughput.

Myelodysplastic syndromes (MDS) are heterogeneous neoplastic disorders of hematopoietic stem cells (HSCs). The current standard of care for patients with MDS is hypomethylating agent (HMA)-based therapy; however, almost 50% of MDS patients fail HMA therapy and progress to acute myeloid leukemia, facing a dismal prognosis due to lack of approved second-line treatment options. As cancer stem cells are the seeds of disease progression, we investigated the biological properties of the MDS HSCs that drive disease evolution, seeking to uncover vulnerabilities that could be therapeutically exploited. Through integrative molecular profiling of HSCs and progenitor cells in large patient cohorts, we found that MDS HSCs in two distinct differentiation states are maintained throughout the clinical course of the disease, and expand at progression, depending on recurrent activation of the anti-apoptotic regulator BCL-2 or nuclear factor-kappa B-mediated survival pathways. Pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden in experimental systems. Further, patients with MDS who progressed after failure to frontline HMA therapy and whose HSCs upregulated BCL-2 achieved improved clinical responses to venetoclax-based therapy in the clinical setting. Overall, our study uncovers that HSC architectures in MDS are potential predictive biomarkers to guide second-line treatments after HMA failure. These findings warrant further investigation of HSC-specific survival pathways to identify new therapeutic targets of clinical potential in MDS. Extensive characterization of the stem and progenitor cell hierarchies of myelodysplastic syndromes reveals compensatory survival mechanisms underpinning the failure of hypomethylating agents, and uncovers biomarkers that predict second-line clinical response to venetoclax-based therapy.

Immunotherapy with T-cells expressing bispecific T-cell engagers (ENG T-cells) is a promising approach to improve the outcomes for patients with recurrent/refractory acute myeloid leukemia (AML). However, similar to T-cells expressing chimeric antigen receptors (CARs), their antitumor activity is limited in the setting of chronic antigen stimulation. We therefore set out to explore whether transgenic expression of IL15 improves the effector function of ENG T-cells targeting CD123-positive AML. T-cells expressing CD123-specific ENG (CD123-ENG) ± IL15 were generated by retroviral transduction from peripheral blood T cells from healthy donors or patients with AML. In this study, we characterized in detail the phenotype and effector functions of ENG T-cell populations in vitro and in vivo . IL15-expressing CD123-ENG (CD123-ENG.IL15) T-cells retained their antigen-specificity and effector function in the setting of chronic antigen exposure for more 30 days of coculture with AML blasts in contrast to CD123-ENG T-cells, whose effector function rapidly eroded. Furthermore, CD123-ENG.IL15 T-cells remained in a less differentiated state as judged by a high frequency of naïve/memory stem T-cell-like cells (CD45RA + CCR7 + /CD45RO − CD62L + cells) without evidence of T-cell exhaustion. Single cell cytokine profiling using IsoPlexis revealed enhanced T-cell polyfunctionality of CD123-ENG.IL15 T-cells as judged by effector cytokine production, including, granzyme B, IFN-γ, MIP-1α, perforin, TNF-α, and TNF-β. In vivo , CD123-ENG.IL15 T-cells exhibited superior antigen-specific anti-AML activity and T-cell persistence in both peripheral blood and tissues (BM, spleens, and livers), resulting in a significant survival advantage in one AML xenograft model and two autologous AML PDX models. In conclusion, we demonstrate here that the expansion, persistence, and anti-AML activity of CD123-ENG T-cells can be significantly improved by transgenic expression of IL15, which promotes a naïve/TSCM-like phenotype. However, we also highlight that targeting a single tumor antigen (CD123) can lead to immune escape, reinforcing the need to develop approaches to target multiple antigens. Likewise, our study demonstrates that it is feasible to evaluate autologous T cells in AML PDX models, which will be critical for future preclinical evaluations of next generation AML-redirected T-cell therapies.

Background Pevonedistat is a first-in-class, small molecular inhibitor of NEDD8-activating enzyme that has clinical activity in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Preclinical data suggest synergy of pevonedistat with azacitidine and venetoclax. Methods This single-center, phase 1/2 study evaluated the combination of azacitidine, venetoclax and pevonedistat in older adults with newly diagnosed secondary AML or with MDS or chronic myelomonocytic leukemia (CMML) after failure of hypomethylating agents. Patients received azacitidine 75 mg/m 2 IV on days 1–7, venetoclax at maximum dose of 200-400 mg orally on days 1–21 (AML cohort) or days 1–14 (MDS/CMML cohort) and pevonedistat 20 mg/m 2 IV on days 1, 3 and 5 for up to 24 cycles. The primary endpoints for the phase 2 portion of the study were the CR/CRi rate in the AML cohort and the overall response rate (CR + mCR + PR + HI) in the MDS/CMML cohort. Findings Forty patients were enrolled (32 with AML and 8 with MDS/CMML). In the AML cohort, the median age was 74 years (range 61–86 years), and 27 patients (84%) had at least one adverse risk cyto-molecular feature, including 15 (47%) with a TP53 mutation or MECOM rearrangement; seventeen patients (53%) had received prior therapy for a preceding myeloid disorder. The CR/CRi rate was 66% (CR 50%; CRi 16%), and the median overall survival (OS) was 8.1 months. In the MDS/CMML cohort, 7 patients (87%) were high or very high risk by the IPSS-R. The overall response rate was 75% (CR 13%; mCR with or without HI 50%; HI 13%). The most common grade 3–4 adverse events were infection in 16 patients (35%), febrile neutropenia in 10 patients (25%) and hypophosphatemia in 9 patients (23%). In an exploratory analysis, early upregulation of NOXA expression was observed, with subsequent decrease in MCL-1 and FLIP, findings consistent with preclinical mechanistic studies of pevonedistat. Upregulation of CD36 was observed, which may have contributed to therapeutic resistance. Conclusions The triplet combination of azacitidine, venetoclax and pevonedistat shows encouraging activity in this very poor-risk population of patients with AML, MDS or CMML. Trial registration ClinicalTrials.gov (NCT03862157).

A key challenge in studying organisms and diseases is to detect rare molecular programs and rare cell populations (RCPs) that drive development, differentiation, and transformation. Molecular features such as genes and proteins defining RCPs are often unknown and difficult to detect from unenriched single-cell data, using conventional dimensionality reduction and clustering-based approaches. Here, we propose an unsupervised approach, SCMER (Single-Cell Manifold presERving feature selection), which selects a compact set of molecular features with definitive meanings that preserve the manifold of the data. We applied SCMER in the context of hematopoiesis, lymphogenesis, tumorigenesis, and drug resistance and response. We found that SCMER can identify non-redundant features that sensitively delineate both common cell lineages and rare cellular states. SCMER can be used for discovering molecular features in a high dimensional dataset, designing targeted, cost-effective assays for clinical applications, and facilitating multi-modality integration.

TP53-mutant acute myeloid leukemia (AML) respond poorly to currently available treatments, including venetoclax-based drug combinations and pose a major therapeutic challenge. Analyses of RNA sequencing and reverse phase protein array datasets revealed significantly lower BAX RNA and protein levels in TP53-mutant compared to TP53–wild-type (WT) AML, a finding confirmed in isogenic CRISPR-generated TP53-knockout and -mutant AML. The response to either BCL-2 (venetoclax) or MCL-1 (AMG176) inhibition was BAX-dependent and much reduced in TP53-mutant compared to TP53-WT cells, while the combination of two BH3 mimetics effectively activated BAX, circumventing survival mechanisms in cells treated with either BH3 mimetic, and synergistically induced cell death in TP53-mutant AML and stem/progenitor cells. The BH3 mimetic–driven stress response and cell death patterns after dual inhibition were largely independent of TP53 status and affected by apoptosis induction. Co-targeting, but not individual targeting of BCL-2 and MCL-1 in mice xenografted with TP53-WT and TP53-R248W Molm13 cells suppressed both TP53-WT and TP53-mutant cell growth and significantly prolonged survival. Our results demonstrate that co-targeting BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance to individual BH3 mimetics in TP53-mutant cells, thus shifting cell fate from survival to death in TP53-deficient and -mutant AML. This concept warrants clinical evaluation.

Chronic graft-versus-host disease (cGvHD) remains a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). A number of studies support a role for B cells in the pathogenesis of cGvHD. In this study, we report the presence of an expanded population of CD19+CD21- B cells with features of exhaustion in the peripheral blood of patients with cGvHD. CD21- B cells were significantly increased in patients with active cGvHD compared to patients without cGvHD and healthy controls (median 12.2 versus 2.12 versus 3%, respectively;  < 0.01). Compared with naïve (CD27-CD21+) and classical memory (CD27+CD21+) B cells, CD19+CD21- B cells in cGvHD were CD10 negative, CD27 negative and CD20hi, and exhibited features of exhaustion, including increased expression of multiple inhibitory receptors such as FCRL4, CD22, CD85J, and altered expression of chemokine and adhesion molecules such as CD11c, CXCR3, CCR7, and CD62L. Moreover, CD21- B cells in cGvHD patients were functionally exhausted and displayed poor proliferative response and calcium mobilization in response to B-cell receptor triggering and CD40 ligation. Finally, the frequencies of circulating CD21- B cells correlated with cGvHD severity in patients after HSCT. Our study further characterizes B cells in chronic cGVHD and supports the use of CD21-CD27-CD10- B cell frequencies as a biomarker of disease severity.