Pediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in the bone marrow or blood (BCP acute lymphoblastic leukemia [BCP-ALL]) or are less common in extramedullary tissue (BCP lymphoblastic lymphoma [BCP-LBL]). Although both presentations are similar in morphology and immunophenotype, molecular studies have been virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is due to its rarity and restriction on small, mostly formalin-fixed paraffin-embedded (FFPE) tissues. Here, to our knowledge, we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n = 97). Whole-exome sequencing indicated a mutational spectrum of BCP-LBL, strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations, and gene expression by RNA sequencing revealed that virtually all molecular subtypes originally defined in BCP-ALL are present in BCP-LBL, with only 7% of lymphomas that were not assigned to a subtype. Similar to BCP-ALL, the most frequent subtypes of BCP-LBL were high hyperdiploidy and ETV6::RUNX1. Tyrosine kinase/cytokine receptor rearrangements were detected in 7% of BCP-LBL. These results indicate that genetic subtypes can be identified in BCP-LBL using next-generation sequencing, even in FFPE tissue, and may be relevant to guide treatment.

Multiple myeloma is a plasma cell malignancy that is currently incurable with conventional therapies. Following the success of CD19-targeted chimeric antigen receptor (CAR) T cells in leukemia and lymphoma, CAR T cells targeting B-cell maturation antigen (BCMA) more recently demonstrated impressive activity in relapsed and refractory myeloma patients. However, BCMA-directed therapy can fail due to weak expression of BCMA on myeloma cells, suggesting that novel approaches to better address this antigen-low disease may improve patient outcomes. We hypothesized that engineered secretion of the proinflammatory cytokine interleukin-18 (IL-18) and multiantigen targeting could improve CAR T-cell activity against BCMA-low myeloma. In a syngeneic murine model of myeloma, CAR T cells targeting the myeloma-associated antigens BCMA and B-cell activating factor receptor (BAFF-R) failed to eliminate myeloma when these antigens were weakly expressed, whereas IL-18-secreting CAR T cells targeting these antigens promoted myeloma clearance. IL-18-secreting CAR T cells developed an effector-like T-cell phenotype, promoted interferon-gamma production, reprogrammed the myeloma bone marrow microenvironment through type-I/II interferon signaling, and activated macrophages to mediate antimyeloma activity. Simultaneous targeting of weakly-expressed BCMA and BAFF-R with dual-CAR T cells enhanced T-cell:target-cell avidity, increased overall CAR signal strength, and stimulated antimyeloma activity. Dual-antigen targeting augmented CAR T-cell secretion of engineered IL-18 and facilitated elimination of larger myeloma burdens in vivo. Our results demonstrate that combination of engineered IL-18 secretion and multiantigen targeting can eliminate myeloma with weak antigen expression through distinct mechanisms.

The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Hematopoietic stem cells (HSCs) are instrumental for organismal survival because they are responsible for lifelong production of mature blood lineages in homeostasis and response to external stress. To fulfill their function, HSCs rely on reciprocal interactions with specialized tissue microenvironments, termed HSC niches. From embryonic development to advanced aging, HSCs transition through several hematopoietic organs in which they are supported by distinct extrinsic cues. Here, we describe recent discoveries on how HSC niches collectively adapt to ensure robust hematopoietic function during biological aging and after exposure to acute stress. We also discuss the latest strategies leveraging niche-derived signals to revert aging-associated phenotypes and enhance hematopoietic recovery after myeloablation.

Wiskott-Aldrich syndrome (WAS) is a multifaceted monogenic disorder with a broad disease spectrum and variable disease severity and a variety of treatment options including allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT). No reliable biomarker exists to predict disease course and outcome for individual patients. A total of 577 patients with a WAS variant from 26 countries and a median follow-up of 8.9 years (range, 0.3-71.1), totaling 6118 patient-years, were included in this international retrospective study. Overall survival (OS) of the cohort (censored at HSCT or GT) was 82% (95% confidence interval, 78-87) at age 15 years and 70% (61-80) at 30 years. The type of variant was predictive of outcome: patients with a missense variant in exons 1 or 2 or with the intronic hot spot variant c.559+5G>A (class I variants) had a 15-year OS of 93% (89-98) and a 30-year OS of 91% (86-97), compared with 71% (62-81) and 48% (34-68) in patients with any other variant (class II; P < .0001). The cumulative incidence rates of disease-related complications such as severe bleeding (P = .007), life-threatening infection (P < .0001), and autoimmunity (P = .004) occurred significantly later in patients with a class I variant. The cumulative incidence of malignancy (P = .6) was not different between classes I and II. It confirms the spectrum of disease severity and quantifies the risk for specific disease-related complications. The class of the variant is a biomarker to predict the outcome for patients with WAS.

Platelet α-granules are rich in transforming growth factor β1 (TGF-β1), which is associated with myeloid-derived suppressor cell (MDSC) biology. Responders to thrombopoietin receptor agonists (TPO-RAs) revealed a parallel increase in the number of both platelets and MDSCs. Here, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to establish an active murine model of immune thrombocytopenia (ITP). Subsequently, we demonstrated that TPO-RAs augmented the inhibitory activities of MDSCs by arresting plasma cells differentiation, reducing Fas ligand expression on cytotoxic T cells, and rebalancing T-cell subsets. Mechanistically, transcriptome analysis confirmed the participation of TGF-β/Smad pathways in TPO-RA-corrected MDSCs, which was offset by Smad2/3 knockdown. In platelet TGF-β1-deficient mice, TPO-RA-induced amplification and enhanced suppressive capacity of MDSCs was waived. Furthermore, our retrospective data revealed that patients with ITP achieving complete platelet response showed superior long-term outcomes compared with those who only reach partial response. In conclusion, we demonstrate that platelet TGF-β1 induces the expansion and functional reprogramming of MDSCs via the TGF-β/Smad pathway. These data indicate that platelet recovery not only serves as an end point of treatment response but also paves the way for immune homeostasis in immune-mediated thrombocytopenia.

Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell-derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell-targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor-independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell-targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

Metabolic tumor volume (MTV) assessed using 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography, a measure of tumor burden, is a promising prognostic indicator in large B-cell lymphoma (LBCL). This exploratory analysis evaluated relationships between baseline MTV (categorized as low [median or less] vs high [greater than median]) and clinical outcomes in the phase 3 ZUMA-7 study (NCT03391466). Patients with LBCL relapsed within 12 months of or refractory to first-line chemoimmunotherapy were randomized 1:1 to axicabtagene ciloleucel (axi-cel; autologous anti-CD19 chimeric antigen receptor T-cell therapy) or standard care (2-3 cycles of chemoimmunotherapy followed by high-dose chemotherapy with autologous stem cell transplantation in patients who had a response). All P values are descriptive. Within high- and low-MTV subgroups, event-free survival (EFS) and progression-free survival (PFS) were superior with axi-cel vs standard care. EFS in patients with high MTV (vs low MTV) was numerically shorter with axi-cel and was significantly shorter with standard care. PFS was shorter in patients with high MTV vs low MTV in both the axi-cel and standard-care arms, and median MTV was lower in patients in ongoing response at data cutoff vs others. Median MTV was higher in patients treated with axi-cel who experienced grade ≥3 neurologic events or cytokine release syndrome (CRS) than in patients with grade 1/2 or no neurologic events or CRS, respectively. Baseline MTV less than or equal to median was associated with better clinical outcomes in patients receiving axi-cel or standard care for second-line LBCL. The trial was registered at www.clinicaltrials.gov as #NCT03391466.

Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor-associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.

Inotuzumab ozogamicin (InO) is an antibody-drug conjugate that delivers calicheamicin to CD22-expressing cells. In a retrospective cohort of InO-treated patients with B-cell acute lymphoblastic leukemia, we sought to understand the genomic determinants of the response and resistance to InO. Pre- and post-InO-treated patient samples were analyzed by whole genome, exome, and/or transcriptome sequencing. Acquired CD22 mutations were observed in 11% (3/27) of post-InO-relapsed tumor samples, but not in refractory samples (0/16). There were multiple CD22 mutations per sample and the mechanisms of CD22 escape included epitope loss (protein truncation and destabilization) and epitope alteration. Two CD22 mutant cases were post-InO hyper-mutators resulting from error-prone DNA damage repair (nonhomologous/alternative end-joining repair, or mismatch repair deficiency), suggesting that hypermutation drove escape from CD22-directed therapy. CD22-mutant relapses occurred after InO and subsequent hematopoietic stem cell transplantation (HSCT), suggesting that InO eliminated the predominant clones, leaving subclones with acquired CD22 mutations that conferred resistance to InO and subsequently expanded. Acquired loss-of-function mutations in TP53, ATM, and CDKN2A were observed, consistent with a compromise of the G1/S DNA damage checkpoint as a mechanism for evading InO-induced apoptosis. Genome-wide CRISPR/Cas9 screening of cell lines identified DNTT (terminal deoxynucleotidyl transferase) loss as a marker of InO resistance. In conclusion, genetic alterations modulating CD22 expression and DNA damage response influence InO efficacy. Our findings highlight the importance of defining the basis of CD22 escape and eradication of residual disease before HSCT. The identified mechanisms of escape from CD22-targeted therapy extend beyond antigen loss and provide opportunities to improve therapeutic approaches and overcome resistance. These trials were registered at www.ClinicalTrials.gov as NCT01134575, NCT01371630, and NCT03441061.