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Primary central nervous system lymphoma (PCNSL) is a rare extranodal lymphomatous malignancy that affects the brain, spinal cord, leptomeninges, or vitreoretinal space, without evidence of systemic involvement. The diagnosis of PCNSL requires a high level of suspicion because clinical presentation varies depending upon involved structures. Initiation of treatment is time sensitive for optimal neurologic recovery and disease control. In general, the prognosis of PCNSL has improved significantly over the past few decades, largely as a result of the introduction and widespread use of high-dose methotrexate (MTX) chemotherapy, which is considered the backbone of first-line polychemotherapy treatment. Upon completion of MTX-based treatment, a consolidation strategy is often required to prolong duration of response. Consolidation can consist of radiation, maintenance therapy, nonmyeloablative chemotherapy, or myeloablative treatment followed by autologous stem cell transplant. Unfortunately, even with consolidation, relapse is common, and 5-year survival rates stand at only 30% to 40%. Novel insights into the pathophysiology of PCNSL have identified key mechanisms in tumor pathogenesis, including activation of the B-cell receptor pathway, immune evasion, and a suppressed tumor immune microenvironment. These insights have led to the identification of novel small molecules targeting these aberrant pathways. The Bruton tyrosine kinase inhibitor ibrutinib and immunomodulatory drugs (lenalidomide or pomalidomide) have shown promising clinical response rates for relapsed/refractory PCNSL and are increasingly used for the treatment of recurrent disease. This review provides a discussion of the clinical presentation of PCNSL, the approach to work-up and staging, and an overview of recent advancements in the understanding of the pathophysiology and current treatment strategies for immunocompetent patients.

Modulation of neutrophil recruitment and function is crucial for targeting inflammatory cells to sites of infection to combat invading pathogens while, at the same time, limiting host tissue injury or autoimmunity. The underlying mechanisms regulating recruitment of neutrophils, 1 of the most abundant inflammatory cells, have gained increasing interest over the years. The previously described classical recruitment cascade of leukocytes has been extended to include capturing, rolling, adhesion, crawling, and transmigration, as well as a reverse-transmigration step that is crucial for balancing immune defense and control of remote organ endothelial leakage. Current developments in the field emphasize the importance of cellular interplay, tissue environmental cues, circadian rhythmicity, detection of neutrophil phenotypes, differential chemokine sensing, and contribution of distinct signaling components to receptor activation and integrin conformations. The use of therapeutics modulating neutrophil activation responses, as well as mutations causing dysfunctional neutrophil receptors and impaired signaling cascades, have been defined in translational animal models. Human correlates of such mutations result in increased susceptibility to infections or organ damage. This review focuses on current advances in the understanding of the regulation of neutrophil recruitment and functionality and translational implications of current discoveries in the field with a focus on acute inflammation and sepsis.

Disseminated intravascular coagulation (DIC) is a syndrome triggered by infectious and noninfectious pathologies characterized by excessive generation of thrombin within the vasculature and widespread proteolytic conversion of fibrinogen. Despite diverse clinical manifestations ranging from thrombo-occlusive damage to bleeding diathesis, DIC etiology commonly involves excessive activation of blood coagulation and overlapping dysregulation of anticoagulants and fibrinolysis. Initiation of blood coagulation follows intravascular expression of tissue factor or activation of the contact pathway in response to pathogen-associated or host-derived, damage-associated molecular patterns. The process is further amplified through inflammatory and immunothrombotic mechanisms. Consumption of anticoagulants and disruption of endothelial homeostasis lower the regulatory control and disseminate microvascular thrombosis. Clinical DIC development in patients is associated with worsening morbidities and increased mortality, regardless of the underlying pathology; therefore, timely recognition of DIC is critical for reducing the pathologic burden. Due to the diversity of triggers and pathogenic mechanisms leading to DIC, diagnosis is based on algorithms that quantify hemostatic imbalance, thrombocytopenia, and fibrinogen conversion. Because current diagnosis primarily assesses overt consumptive coagulopathies, there is a critical need for better recognition of nonovert DIC and/or pre-DIC states. Therapeutic strategies for patients with DIC involve resolution of the eliciting triggers and supportive care for the hemostatic imbalance. Despite medical care, mortality in patients with DIC remains high, and new strategies, tailored to the underlying pathologic mechanisms, are needed.

Autoimmune conditions can occur in a temporary relationship with any malignant lymphoma. In many instances, treatment at diagnosis is not required, but symptomatic autoimmune conditions represent an indication for treatment, particularly in chronic lymphoproliferative diseases. Treatment is selected depending on the predominant condition: autoimmune disease (immunosuppression) or lymphoma (antilymphoma therapy). Steroids and anti-CD20 antibodies are effective against both conditions and may suppress the autoimmune complication for a prolonged period. The efficacy of B-cell receptor inhibitors has provided us with novel insights into the pathophysiology of antibody-producing B cells. Screening for underlying autoimmune conditions is part of the lymphoma workup, because other drugs, such as immunomodulators and checkpoint inhibitors, should be avoided or used with caution. In this article, we discuss diagnostic challenges and treatment approaches for different situations involving lymphomas and autoimmune cytopenias.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild-type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.

CD19-directed chimeric antigen receptor–modified (CAR T) T cells achieve durable remissions in about 30% to 40% of relapsed/refractory large B-cell lymphomas. T-cell exhaustion and/or an immunosuppressive tumor microenvironment may contribute to CAR T-cell failure. Pembrolizumab, an anti-PD1 immune checkpoint inhibitor, may reverse T-cell exhaustion after CAR T-cell therapy. We treated 12 patients with B-cell lymphomas who were either refractory to (n = 9) or relapsed after (n = 3) CD19-directed CAR T-cell (4-1BB–costimulated) therapy with pembrolizumab 200 mg IV every 3 weeks. Median time from CAR T-cell infusion to first pembrolizumab dose was 3.3 months (range, 0.4-42.8 months). Pembrolizumab was well tolerated, and the only grade ≥3 adverse events related to pembrolizumab were neutropenia (n = 3; 25%). Best overall response rate after pembrolizumab was 25% (3 of 12 patients; 1 complete response; 2 partial responses). One (8%) patient had stable disease; thus, 4 of 12 (33%) patients had clinical benefit. After pembrolizumab, 4 patients with clinical benefit had an increase in percentage of CAR T cells by mass cytometry by time of flight (CyTOF); 3 of 4 of these patients also had increases in CAR19 transgene levels by quantitative polymerase chain reaction. Deep immune profiling using CyTOF revealed increased CAR T-cell activation and proliferation and less T-cell exhaustion in clinical responders. Together, PD1 blockade with pembrolizumab after CD19-directed CAR T-cell therapy appears safe and may achieve clinical responses in some patients with B-cell lymphomas refractory to or relapsed after CAR T-cell therapy. This trial was registered at www.clinicaltrials.gove as #NCT02650999.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform–specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3′-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell–like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1−/− accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1−/− (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21–encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

YTHDC1 has distinct functions as a nuclear N6-methyladenosine (m6A) reader in regulating RNA metabolism. Here we show that YTHDC1 is overexpressed in acute myeloid leukemia (AML) and that it is required for the proliferation and survival of human AML cells. Genetic deletion of Ythdc1 markedly blocks AML development and maintenance as well as self-renewal of leukemia stem cells (LSCs) in vivo in mice. We found that Ythdc1 is also required for normal hematopoiesis and hematopoietic stem and progenitor cell (HSPC) maintenance in vivo. Notably, Ythdc1 haploinsufficiency reduces self-renewal of LSCs but not HSPCs in vivo. YTHDC1 knockdown has a strong inhibitory effect on proliferation of primary AML cells. Mechanistically, YTHDC1 regulates leukemogenesis through MCM4, which is a critical regulator of DNA replication. Our study provides compelling evidence that shows an oncogenic role and a distinct mechanism of YTHDC1 in AML.

Rapid advances in large-scale next-generation sequencing studies of human samples have progressively defined the highly heterogeneous genetic landscape of chronic lymphocytic leukemia (CLL). At the same time, the numerous challenges posed by the difficulties in rapid manipulation of primary B cells and the paucity of CLL cell lines have limited the ability to interrogate the function of the discovered putative disease “drivers,” defined in human sequencing studies through statistical inference. Mouse models represent a powerful tool to study mechanisms of normal and malignant B-cell biology and for preclinical testing of novel therapeutics. Advances in genetic engineering technologies, including the introduction of conditional knockin/knockout strategies, have opened new opportunities to model genetic lesions in a B-cell–restricted context. These new studies build on the experience of generating the MDR mice, the first example of a genetically faithful CLL model, which recapitulates the most common genomic aberration of human CLL: del(13q). In this review, we describe the application of mouse models to the studies of CLL pathogenesis and disease transformation from an indolent to a high-grade malignancy (ie, Richter syndrome [RS]) and treatment, with a focus on newly developed genetically inspired mouse lines modeling recurrent CLL genetic events. We discuss how these novel mouse models, analyzed using new genomic technologies, allow the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible and provide important insight into human CLL and RS pathogenesis and therapeutic vulnerabilities. These models thereby provide valuable platforms for functional genomic analyses and treatment studies.

Proper regulation of p53 signaling is critical for the maintenance of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). The hematopoietic cell–specific mechanisms regulating p53 activity remain largely unknown. Here, we demonstrate that conditional deletion of acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) in hematopoietic cells impairs repopulation capacity and postinjury regeneration of HSCs. Mechanistically, ANP32B forms a repressive complex with p53 and thus inhibits the transcriptional activity of p53 in hematopoietic cells, and p53 deletion rescues the functional defect in Anp32b-deficient HSCs. Of great interest, ANP32B is highly expressed in leukemic cells from patients with chronic myelogenous leukemia (CML). Anp32b deletion enhances p53 transcriptional activity to impair LSC function in a murine CML model and exhibits synergistic therapeutic effects with tyrosine kinase inhibitors in inhibiting CML propagation. In summary, our findings provide a novel strategy to enhance p53 activity in LSCs by inhibiting ANP32B and identify ANP32B as a potential therapeutic target in treating CML.

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm with a heterogeneous clinical and biological behavior. SOX11 oncogenic expression contributes to the aggressiveness of these tumors by different mechanisms, including tumor and stromal cell interactions. However, the precise composition of the immune cell microenvironment of MCL, its possible relationship to SOX11 expression, and how it may contribute to tumor behavior is not well known. Here, we performed an integrative transcriptome analysis of 730 immune-related genes combined with the immune cell phenotype analysis by immunohistochemistry in SOX11+ and SOX11− primary nodal MCL cases and non-neoplastic reactive lymph nodes. SOX11+ MCL had a significant lower T-cell intratumoral infiltration compared with negative cases. A reduced expression of MHCI/II-like and T-cell costimulation and signaling activation related transcripts was significantly associated with poor clinical outcome. Moreover, we identified CD70 as a SOX11 direct target gene, whose overexpression was induced in SOX11+, but not SOX11− tumor cells by CD40L in vitro. CD70 was overexpressed in primary SOX11+ MCL and it was associated with an immune unbalance of the tumor microenvironment characterized by increased number of effector regulatory T (Treg) cell infiltration, higher proliferation, and aggressive clinical course. CD27 was expressed with moderate to strong intensity in 76% of cases. Overall, our results suggest that SOX11 expression in MCL is associated with an immunosuppressive microenvironment characterized by CD70 overexpression in tumor cells, increased Treg cell infiltration and downmodulation of antigen processing, and presentation and T-cell activation that could promote MCL progression and represent a potential target for tailored therapies.

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.

t(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in the infant and pediatric population, yet we have little information on the molecular mechanisms responsible for disease progression. This impairs the development of therapeutic regimens that can address the aggressive phenotype and lineage plasticity of MLL-AF4–driven leukemogenesis. This study highlights novel mechanisms of disease development by focusing on 2 microRNAs (miRNAs) upregulated in leukemic blasts from primary patient samples: miR-130b and miR-128a. We show that miR-130b and miR-128a are downstream targets of MLL-AF4 and can individually drive the transition from a pre-leukemic stage to an acute leukemia in an entirely murine Mll-AF4 in vivo model. They are also required to maintain the disease phenotype. Interestingly, miR-130b overexpression led to a mixed/B-cell precursor (BCP)/myeloid leukemia, propagated by the lymphoid-primed multipotent progenitor (LMPP) population, whereas miR-128a overexpression resulted in a pro-B acute lymphoblastic leukemia (ALL), maintained by a highly expanded Il7r+c-Kit+ blast population. Molecular and phenotypic changes induced by these two miRNAs fully recapitulate the human disease, including central nervous system infiltration and activation of an MLL-AF4 expression signature. Furthermore, we identified 2 downstream targets of these miRNAs, NR2F6 and SGMS1, which in extensive validation studies are confirmed as novel tumor suppressors of MLL-AF4+ leukemia. Our integrative approach thus provides a platform for the identification of essential co-drivers of MLL-rearranged leukemias, in which the preleukemia to leukemia transition and lineage plasticity can be dissected and new therapeutic approaches can be tested.

Immunoglobulin M (IgM) multiple myeloma (MM) is a rare disease subgroup. Its differentiation from other IgM-producing gammopathies such as Waldenström macroglobulinemia (WM) has not been well characterized but is essential for proper risk assessment and treatment. In this study, we investigated genomic and transcriptomic characteristics of IgM-MM samples using whole-genome and transcriptome sequencing to identify differentiating characteristics from non–IgM-MM and WM. Our results suggest that IgM-MM shares most of its defining structural variants and gene-expression profiling with MM, but has some key characteristics, including t(11;14) translocation, chromosome 6 and 13 deletion as well as distinct molecular and transcription-factor signatures. Furthermore, IgM-MM translocations were predominantly characterized by VHDHJH recombination-induced breakpoints, as opposed to the usual class-switching region breakpoints; coupled with its lack of class switching, these data favor a pre–germinal center origin. Finally, we found elevated expression of clinically relevant targets, including CD20 and Bruton tyrosine kinase, as well as high BCL2/BCL2L1 ratio in IgM-MM, providing potential for targeted therapeutics.

Activating mutations in MYD88 promote malignant cell growth and survival through hematopoietic cell kinase (HCK)–mediated activation of Bruton tyrosine kinase (BTK). Ibrutinib binds to BTKCys481 and is active in B-cell malignancies driven by mutated MYD88. Mutations in BTKCys481, particularly BTKCys481Ser, are common in patients with acquired ibrutinib resistance. We therefore performed an extensive medicinal chemistry campaign and identified KIN-8194 as a novel dual inhibitor of HCK and BTK. KIN-8194 showed potent and selective in vitro killing of MYD88-mutated lymphoma cells, including ibrutinib-resistant BTKCys481Ser-expressing cells. KIN-8194 demonstrated excellent bioavailability and pharmacokinetic parameters, with good tolerance in rodent models at pharmacologically achievable and active doses. Pharmacodynamic studies showed sustained inhibition of HCK and BTK for 24 hours after single oral administration of KIN-8194 in an MYD88-mutated TMD-8 activated B-cell diffuse large B-cell lymphoma (ABC DLBCL) and BCWM.1 Waldenström macroglobulinemia (WM) xenografted mice with wild-type BTK (BTKWT)– or BTKCys481Ser-expressing tumors. KIN-8194 showed superior survival benefit over ibrutinib in both BTKWT- and BTKCys481Ser-expressing TMD-8 DLBCL xenografted mice, including sustained complete responses of >12 weeks off treatment in mice with BTKWT-expressing TMD-8 tumors. The BCL_2 inhibitor venetoclax enhanced the antitumor activity of KIN-8194 in BTKWT- and BTKCys481Ser-expressing MYD88-mutated lymphoma cells and markedly reduced tumor growth and prolonged survival in mice with BTKCys481Ser-expressing TMD-8 tumors treated with both drugs. The findings highlight the feasibility of targeting HCK, a key driver of mutated MYD88 pro-survival signaling, and provide a framework for the advancement of KIN-8194 for human studies in B-cell malignancies driven by HCK and BTK.

Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.

Recent studies have shown that maternal anti-CD36 antibodies represent a frequent cause of fetal/neonatal alloimmune thrombocytopenia (FNAIT) in Asian and African populations. However, little is known about the pathomechanism and antenatal treatment of anti-CD36–mediated FNAIT. Here, we established a novel animal model to examine the clinical features of pups from immunized Cd36−/− female mice after breeding with wild-type male mice. Mild thrombocytopenia was observed, but high pup mortality was also documented (40.26%). Administration of intravenous immunoglobulin (IVIG) (1 g/kg) on days 7, 12, and 17 to immunized Cd36−/− mothers after breeding reduced fetal death (12.70%). However, delaying the IVIG administration series on days 10, 15, and 20 did not reduce fetal death (40.00%). In contrast, injection of deglycosylated anti-CD36 (deg-anti-CD36) polyclonal antibodies (5 mg/kg) on days 10, 15, and 20 significantly reduced fetal death (5.26%). Subsequently, monoclonal antibodies (mAbs) against mouse CD36 were developed, and one clone producing high-affinity anti-CD36 (termed 32-106) effectively inhibited maternal antibody binding and was therefore selected. Using the same approach of deg-anti-CD36, the administration of deg-32-106 significantly reduced fetal death (2.17%). Furthermore, immunized Cd36−/− mothers exhibited placental deficiency. Accordingly, maternal anti-CD36 antibodies inhibited angiogenesis of placenta endothelial cells, which could be restored by deg-32-106. In summary, maternal anti-CD36 antibodies caused a high frequency of fetal death in our animal model, associated with placental dysfunction. This deleterious effect could be diminished by the antenatal administration of IVIG and deg-mAb 32-106. Interestingly, treatment with deg-32-106 seems more beneficial considering the lower dose, later start of treatment, and therapy success.

MYD88 and CXCR4 mutations are common in Waldenström macroglobulinemia (WM). Mutated CXCR4 (CXCR4Mut) impacts BTK-inhibitor response. We conducted a phase 1 trial of the CXCR4-antagonist ulocuplumab with ibrutinib in this first-ever study to target CXCR4Mut in WM. Ibrutinib was initiated at 420 mg/d with cycle 1 and continued until intolerance or progression; ulocuplumab was given cycles 1 to 6, with a 3 + 3 dose-escalation design. Each cycle was 4 weeks. Thirteen symptomatic patients, of whom 9 were treatment-naive patients were enrolled. Twelve were evaluable for response. At best response, their median serum immunoglobulin M declined from 5574 to 1114 mg/dL; bone marrow disease decreased from 65% to 10%, and hemoglobin increased from 10.1 to 14.2 g/dL (P < .001). The major and VGPR response rates were 100% and 33%, respectively, with VGPRs observed at lower ulocuplumab dose cohorts. Median times to minor and major responses were 0.9 and 1.2 months, respectively. With a median follow-up of 22.4 months, the estimated 2-year progression-free survival was 90%. The most frequent recurring grade ≥2 adverse events included reversible thrombocytopenia, rash, and skin infections. Ulocuplumab dose-escalation did not impact adverse events. The study demonstrates the feasibility of combining a CXCR4-antagonist with ibrutinib and provides support for the development of CXCR4-antagonists for CXCR4Mut WM. This trial was registered at www.clinicaltrials.gov as #NCT03225716.

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter–driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell–derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene–modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

MYB plays a key role in gene regulation throughout the hematopoietic hierarchy and is critical for the maintenance of normal hematopoietic stem cells (HSC). Acquired genetic dysregulation of MYB is involved in the etiology of a number of leukemias, although inherited noncoding variants of the MYB gene are a susceptibility factor for many hematological conditions, including myeloproliferative neoplasms (MPN). The mechanisms that connect variations in MYB levels to disease predisposition, especially concerning age dependency in disease initiation, are completely unknown. Here, we describe a model of Myb insufficiency in mice that leads to MPN, myelodysplasia, and leukemia in later life, mirroring the age profile of equivalent human diseases. We show that this age dependency is intrinsic to HSC, involving a combination of an initial defective cellular state resulting from small effects on the expression of multiple genes and a progressive accumulation of further subtle changes. Similar to previous studies showing the importance of proteostasis in HSC maintenance, we observed altered proteasomal activity and elevated proliferation indicators, followed by elevated ribosome activity in young Myb-insufficient mice. We propose that these alterations combine to cause an imbalance in proteostasis, potentially creating a cellular milieu favoring disease initiation.

Bruton tyrosine kinase inhibitors (BTKi) have significantly changed the treatment landscape for patients with B-cell malignancies, including chronic lymphocytic leukemia, Waldenstrom macroglobulinemia, mantle cell lymphoma, and marginal zone lymphoma. Unfortunately, patients with BTKi-resistant disease have shortened survival. Clinical and molecular risk factors, such as number of prior therapies and presence of TP53 mutations, can be used to predict patients at the highest risk of developing BTKi resistance. Many mechanisms of BTKi resistance have been reported with mutations in BTK and phospholipase C γ2 supported with the most data. The introduction of venetoclax has lengthened the survival of patients with BTKi-resistant disease. Ongoing clinical trials with promising treatment modalities, such as next-generation BTKi and chimeric antigen receptor T-cell therapy, have reported promising efficacy in patients with BTKi-resistant disease. Continued research focusing on resistance mechanisms and methods of how to circumvent resistance is needed to further prolong the survival of patients with BTKi-resistant B-cell malignancies.

Most patients with acute promyelocytic leukemia (APL) can be cured with combined all-trans retinoic acid (ATRA) and arsenic trioxide therapy, which induces the destruction of PML-RARA, the initiating fusion protein for this disease. However, the underlying mechanisms by which PML-RARA initiates and maintains APL cells are still not clear. Therefore, we identified genes that are dysregulated by PML-RARA in mouse and human APL cells and prioritized GATA2 for functional studies because it is highly expressed in preleukemic cells expressing PML-RARA, its high expression persists in transformed APL cells, and spontaneous somatic mutations of GATA2 occur during APL progression in mice and humans. These and other findings suggested that GATA2 may be upregulated to thwart the proliferative signal generated by PML-RARA and that its inactivation by mutation (and/or epigenetic silencing) may accelerate disease progression in APL and other forms of acute myeloid leukemia (AML). Indeed, biallelic knockout of Gata2 with CRISPR/Cas9-mediated gene editing increased the serial replating efficiency of PML-RARA–expressing myeloid progenitors (as well as progenitors expressing RUNX1-RUNX1T1, or deficient for Cebpa), increased mouse APL penetrance, and decreased latency. Restoration of Gata2 expression suppressed PML-RARA–driven aberrant self-renewal and leukemogenesis. Conversely, addback of a mutant GATA2R362G protein associated with APL and AML minimally suppressed PML-RARA–induced aberrant self-renewal, suggesting that it is a loss-of-function mutation. These studies reveal a potential role for Gata2 as a tumor suppressor in AML and suggest that restoration of its function (when inactivated) may provide benefit for AML patients.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous diagnostic category comprising distinct molecular subtypes characterized by diverse genetic aberrations that dictate patient outcome. As roughly one-third of patients with DLBCL are not cured by current standard chemoimmunotherapy, a better understanding of the molecular pathogenesis is warranted to improve outcome. B-cell receptor (BCR) signaling is crucial for the development, growth, and survival of normal B cells and a substantial fraction of malignant B cells. Various analyses revealed genetic alterations of central components of the BCR or its downstream signaling effectors in some subtypes of DLBCL. Thus, BCR signaling and the downstream NF-κB and phosphatidylinositol 3-kinase (PI3K) cascades have been proposed as potential targets for the treatment of patients with DLBCL. As one of the main effectors of BCR activation, PI3K-mediated signals play a crucial role in the pathogenesis and survival of DLBCL. In this review, we summarize our current understanding of BCR signaling with a special focus on the PI3K pathway in DLBCL and how to use this knowledge therapeutically.

Following a similar report on multiple myeloma, Ghione and colleagues report the expected observation that patients with non-Hodgkin lymphoma (NHL) receiving anti-B cell therapies have markedly reduced antibody responses to COVID-19 immunization. Although there is no information regarding T-cell immunity, this suggests that while vaccination is certainly still recommended for this population, patients should be strongly encouraged to maintain social distancing precautions and should be revaccinated after an appropriate interval from the end of their antilymphoma therapy.

Nongenetic heterogeneity, or gene expression stochasticity, is an important source of variability in biological systems. With the advent and improvement of single molecule resolution technologies, it has been shown that transcription dynamics and resultant transcript number fluctuations generate significant cell-to-cell variability that has important biological effects and may contribute substantially to both tissue homeostasis and disease. In this respect, the pathophysiology of stem cell-derived malignancies such as acute myeloid leukemia and myelodysplastic syndromes, which has historically been studied at the ensemble level, may require reevaluation. To that end, it is our aim in this review to highlight the results of recent single-molecule, biophysical, and systems studies of gene expression dynamics, with the explicit purpose of demonstrating how the insights from these basic science studies may help inform and progress the field of leukemia biology and, ultimately, research into novel therapies.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL), typically to an aggressive lymphoma. Treatment options for RS are limited and the disease is often fatal. Receptor tyrosine kinase–like orphan receptor 1 (ROR1) is expressed on CLL cells and other cancers but not on healthy adult tissues, making it an attractive, tumor-specific therapeutic target. VLS-101 is being developed as an antibody-drug conjugate (ADC) for therapy of ROR1-expressing (ROR1+) cancers. VLS-101 comprises UC-961 (a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE). VLS-101 binding to ROR1 results in rapid cellular internalization and delivery of MMAE to induce tumor cell death. We studied 4 RS patient-derived xenografts (RS-PDXs) with varying levels of ROR1 expression (11%, 32%, 85%, and 99% of cells). VLS-101 showed no efficacy in the lowest-expressing RS-PDX but induced complete remissions in those with higher levels of ROR1 expression. Responses were maintained during the posttherapy period, particularly after higher VLS-101 doses. In systemic ROR1+ RS-PDXs, VLS-101 dramatically decreased tumor burden in all RS-colonized tissues and significantly prolonged survival. Animals showed no adverse effects or weight loss. Our results confirm ROR1 as a target in RS and demonstrate the therapeutic potential of using an ADC directed toward ROR1 for the treatment of hematological cancers. A phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with RS and other hematological malignancies.

The bone marrow (BM) is responsible for generating and maintaining lifelong output of blood and immune cells. In addition to its key hematopoietic function, the BM acts as an important lymphoid organ, hosting a large variety of mature lymphocyte populations, including B cells, T cells, natural killer T cells, and innate lymphoid cells. Many of these cell types are thought to visit the BM only transiently, but for others, like plasma cells and memory T cells, the BM provides supportive niches that promote their long-term survival. Interestingly, accumulating evidence points toward an important role for mature lymphocytes in the regulation of hematopoietic stem cells (HSCs) and hematopoiesis in health and disease. In this review, we describe the diversity, migration, localization, and function of mature lymphocyte populations in murine and human BM, focusing on their role in immunity and hematopoiesis. We also address how various BM lymphocyte subsets contribute to the development of aplastic anemia and immune thrombocytopenia, illustrating the complexity of these BM disorders and the underlying similarities and differences in their disease pathophysiology. Finally, we summarize the interactions between mature lymphocytes and BM resident cells in HSC transplantation and graft-versus-host disease. A better understanding of the mechanisms by which mature lymphocyte populations regulate BM function will likely improve future therapies for patients with benign and malignant hematologic disorders.

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor–targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide–DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, β2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node–derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton’s tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.

In chronic lymphocytic leukemia (CLL), increasing knowledge of the biology of the tumor cells has led to transformative improvements in our capacity to assess and treat patients. The dependence of tumor cells on surface immunoglobulin receptor signaling, survival pathways, and accessory cells within the microenvironment has led to a successful double-barreled attack with designer drugs. Studies have revealed that CLL should be classified based on the mutational status of the expressed IGHV sequences into 2 diseases, either unmutated (U) or mutated (M) CLL, each with a distinctive cellular origin, biology, epigenetics/genetics, and clinical behavior. The origin of U-CLL lies among the natural antibody repertoire, and dominance of IGHV1-69 reveals a superantigenic driver. In both U-CLL and M-CLL, a calibrated stimulation of tumor cells by self-antigens apparently generates a dynamic reiterative cycle as cells, protected from apoptosis, transit between blood and tissue sites. But there are differences in outcome, with the balance between proliferation and anergy favoring anergy in M-CLL. Responses are modulated by an array of microenvironmental interactions. Availability of T-cell help is a likely determinant of cell fate, the dependency on which varies between U-CLL and M-CLL, reflecting the different cells of origin, and affecting clinical behavior. Despite such advances, cell-escape strategies, Richter transformation, and immunosuppression remain as challenges, which only may be met by continued research into the biology of CLL.

Tumor heterogeneity complicates biomarker development and fosters drug resistance in solid malignancies. In lymphoma, our knowledge of site-to-site heterogeneity and its clinical implications is still limited. Here, we profiled 2 nodal, synchronously acquired tumor samples from 10 patients with follicular lymphoma (FL) using single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing, and flow cytometry. By following the rapidly mutating tumor immunoglobulin genes, we discovered that BCR subclones were shared between the 2 tumor sites in some patients, but in many patients, the disease had evolved separately with limited tumor cell migration between the sites. Patients exhibiting divergent BCR evolution also exhibited divergent tumor gene-expression and cell-surface protein profiles. While the overall composition of the tumor microenvironment did not differ significantly between sites, we did detect a specific correlation between site-to-site tumor heterogeneity and T follicular helper (Tfh) cell abundance. We further observed enrichment of particular ligand-receptor pairs between tumor and Tfh cells, including CD40 and CD40LG, and a significant correlation between tumor CD40 expression and Tfh proliferation. Our study may explain discordant responses to systemic therapies, underscores the difficulty of capturing a patient’s disease with a single biopsy, and furthers our understanding of tumor-immune networks in FL.

Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

Serum and glucocorticoid-regulated kinase 1 (SGK1) is one of the most frequently mutated genes in diffuse large B-cell lymphoma (DLBCL). However, little is known about its function or the consequence of its mutation. The frequent finding of truncating mutations has led to the widespread assumption that these represent loss-of-function variants and, accordingly, that SGK1 must act as a tumor suppressor. In this study, instead, the most common SGK1 mutations led to production of aberrantly spliced messenger RNA neoisoforms in which translation is initiated from downstream methionines. The resulting N-terminal truncated protein isoforms showed increased expression related to the exclusion of an N-terminal degradation domain. However, they retained a functional kinase domain, the overexpression of which rendered cells resistant to AKT inhibition, in part because of increased phosphorylation of GSK3B. These findings challenge the prevailing assumption that SGK1 is a tumor-suppressor gene in DLBCL and provide the impetus to explore further the pharmacological inhibition of SGK1 as a therapeutic strategy for DLBCL.

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII, and biochemical studies have suggested APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein with an APC-resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately fivefold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, a tail clip assay was performed in homozygous HA/FV Leiden (FVL) mice infused with FVIII-QQ or FVIII-WT in the presence or absence of monoclonal antibody 1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FVL mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, indicating the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics.

Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy for which there is an unmet need for novel treatment strategies. Here, we characterize the growth arrest and DNA damage-inducible gene gamma (GADD45g) as a novel tumor suppressor in AML. We show that GADD45g is preferentially silenced in AML, especially in AML with FMS-like tyrosine kinase 3–internal tandem duplication (FLT3-ITD) mutations and mixed-lineage leukemia (MLL)-rearrangements, and reduced expression of GADD45g is correlated with poor prognosis in patients with AML. Upregulation of GADD45g impairs homologous recombination DNA repair, leading to DNA damage accumulation, and dramatically induces apoptosis, differentiation, and growth arrest and increases sensitivity of AML cells to chemotherapeutic drugs, without affecting normal cells. In addition, GADD45g is epigenetically silenced by histone deacetylation in AML, and its expression is further downregulated by oncogenes FLT3-ITD and MLL-AF9 in patients carrying these genetic abnormalities. Combination of the histone deacetylase 1/2 inhibitor romidepsin with the FLT3 tyrosine kinase inhibitor AC220 or the bromodomain inhibitor JQ1 exerts synergistic antileukemic effects on FLT3-ITD+ and MLL-AF9+ AML, respectively, by dually activating GADD45g. These findings uncover hitherto unreported evidence for the selective antileukemic role of GADD45g and provide novel strategies for the treatment of FLT3-ITD+ and MLL-AF9+ AML.

The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.

High coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.

Apoptosis induction by death receptor (DR)-specific agonistic antibodies is a potentially effective antitumor therapy. Nonetheless, to date, all conventional DR-targeting antibodies that induce apoptosis via FcγR-dependent DR clustering failed to show clinical efficacy. HexaBody-DR5/DR5 (GEN1029) has been developed to overcome full FcγR dependence. HexaBody-DR5/DR5 is a mixture of 2 noncompeting DR5-specific immunoglobulin G1 (IgG1) antibodies, each with an E430G mutation in the Fc domain. This mutation enhances Fc-Fc interactions, resulting in antibody hexamerization, followed by FcγR-independent clustering of DR5 molecules. This unique combination of dual epitope targeting and increased IgG hexamerization resulted in potent preclinical antitumor activity in various solid cancers. In this study, we explored the preclinical activity of HexaBody-DR5/DR5 in multiple myeloma (MM), because MM cells are known to express DR5. In bone marrow samples from 48 MM patients, HexaBody-DR5/DR5 induced potent cytotoxicity of primary MM cells. Importantly, HexaBody-DR5/DR5 mediated the highest cytotoxic activity in samples from relapsed/refractory MM patients, including those who are refractory to daratumumab. This improved cytotoxic activity was observed only in patients who received their last anti-MM treatment

We have recently discovered Japanese children with a novel Fanconi anemia–like inherited bone marrow failure syndrome (IBMFS). This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. Phytohemagglutinin-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here, we report that, in contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate various amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including induced pluripotent stem cells (iPSCs). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde, as indicated by higher levels of DNA damage than in FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.

The KIT D816V mutation is found in >80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. Therefore, KIT D816V represents a prime therapeutic target for SM. Here, we generated a panel of patient-specific KIT D816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM and mast cell leukemia to develop a patient-specific SM disease model for mechanistic and drug-discovery studies. KIT D816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KIT D816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KIT D816V iPSC hematopoiesis. KIT D816V causes constitutive activation of the KIT tyrosine kinase receptor, and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib, a US Food and Drug Administration–approved angiokinase inhibitor that targets vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KIT D816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the adenosine triphosphate binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V–targeted therapy of advanced SM.

Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies.

Plasma cells no longer express a B-cell antigen receptor and are hence deprived of signals crucial for survival throughout B-cell development. Instead, normal plasma cells, as well as their malignant myeloma counterparts, heavily rely on communication with the bone marrow (BM) microenvironment for survival. The plasma cell heparan sulfate proteoglycan (HSPG) syndecan-1 (CD138) and HSPGs in the BM microenvironment act as master regulators of this communication by co-opting specific growth and survival factors from the BM niche. This designates syndecan-1/HSPGs and their synthesis machinery as potential treatment targets in multiple myeloma.

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4(+) T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter–driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell–derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene–modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.