PI3Kδ/γ inhibition promotes human CART cell epigenetic and metabolic reprogramming to enhance antitumor cytotoxicity.

Current limitations in using chimeric antigen receptor T(CART) cells to treat patients with hematological cancers include limited expansion and persistence in vivo that contribute to cancer relapse. Patients with chronic lymphocytic leukemia (CLL) have terminally differentiated T cells with an exhausted phenotype and experience low complete response rates after autologous CART therapy. Because PI3K inhibitor therapy is associated with the development of T-cell-mediated autoimmunity, we studied the effects of inhibiting the PI3Kδ and PI3Kγ isoforms during the manufacture of CART cells prepared from patients with CLL. Dual PI3Kδ/γ inhibition normalized CD4/CD8 ratios and maximized the number of CD8+ T-stem cell memory, naive, and central memory T-cells with dose-dependent decreases in expression of the TIM-3 exhaustion marker. CART cells manufactured with duvelisib (Duv-CART cells) showed significantly increased in vitro cytotoxicity against CD19+ CLL targets caused by increased frequencies of CD8+ CART cells. Duv-CART cells had increased expression of the mitochondrial fusion protein MFN2, with an associated increase in the relative content of mitochondria. Duv-CART cells exhibited increased SIRT1 and TCF1/7 expression, which correlated with epigenetic reprograming of Duv-CART cells toward stem-like properties. After transfer to NOG mice engrafted with a human CLL cell line, Duv-CART cells expressing either a CD28 or 41BB costimulatory domain demonstrated significantly increased in vivo expansion of CD8+ CART cells, faster elimination of CLL, and longer persistence. Duv-CART cells significantly enhanced survival of CLL-bearing mice compared with conventionally manufactured CART cells. In summary, exposure of CART to a PI3Kδ/γ inhibitor during manufacturing enriched the CART product for CD8+ CART cells with stem-like qualities and enhanced efficacy in eliminating CLL in vivo.

Multicolor Light-Induced Immune Activation via Polymer Photocaged Cytokines.

Cytokines act as potent, extracellular signals of the human immune system and can elicit striking treatment responses in patients with autoimmune disease, tissue damage, and cancer. Yet, despite their therapeutic potential, recombinant cytokine-mediated immune responses remain difficult to control as their administration is often systemic, whereas their intended sites of action are localized. To address the challenge of spatially and temporally constraining cytokine signals, we recently devised a strategy whereby recombinant cytokines are reversibly inactivated via chemical modification with photo-labile polymers that respond to visible LED light. Extending this approach to enable both in vivo and multicolor immune activation, here we describe a strategy whereby cytokines appended with heptamethine cyanine-polyethylene glycol are selectively re-activated ex vivo using tissue-penetrating near-infrared (NIR) light. We show that NIR LED light illumination of caged, pro-inflammatory cytokines restores cognate receptor signaling and potentiates the activity of T cell-engager cancer immunotherapies ex vivo. Using combinations of visible- and NIR-responsive cytokines, we further demonstrate multiwavelength optical control of T cell cytolysis ex vivo, as well as the ability to perform Boolean logic using multicolored light and orthogonally photocaged cytokine pairs as inputs and T cell activity as outputs. Together, this work demonstrates a novel approach to control extracellular immune cell signals using light, a strategy that in the future may improve our understanding of and ability to treat cancer and other diseases.

Bortezomib enhances cytotoxicity of ex vivo-expanded gamma delta T cells against acute myeloid leukemia and T-cell acute lymphoblastic leukemia.

Engagement between the natural killer group 2, member D (NKG2D) receptor and its ligands is one of the main mechanisms used by immune cells to target stressed cells for cell death. NKG2D ligands are known markers of cellular stress and are often upregulated on tumor cells. Certain drugs can further increase NKG2D ligand levels, thereby making tumor cells more susceptible to immune cell detection and destruction. However, the effectiveness of this approach appears to be limited with drug treatment alone, possibly due to immune dysregulation in the setting of malignancies. We hypothesized that a more effective approach would be a combination of NKG2D ligand-inducing drugs, such as the proteasome inhibitor bortezomib, and ex vivo-expanded peripheral blood γδ T cells (i.e., Vγ9Vδ2 T cells). Acute myeloid leukemia (AML) is a high-risk hematologic malignancy, and treatment has shown limited benefit with the addition of bortezomib to standard chemotherapy regimens. Two AML cells lines, Nomo-1 and Kasumi-1, were treated with increasing concentrations of bortezomib, and changes in NKG2D ligand expression were measured. Bortezomib treatment significantly increased expression of the NKG2D ligand UL16 binding protein (ULBP) 2/5/6 in both cell lines. Vγ9Vδ2 T cells were expanded and isolated from peripheral blood of healthy donors to generate a final cellular product with a mean of 96% CD3+/γδ T-cell receptor-positive cells. Combination treatment of the AML cell lines with γδ T cells and bortezomib resulted in significantly greater cytotoxicity than γδ T cells alone, even at lower effector-to-target ratios. Based on the positive results against AML and the generalizable mechanism of this combination approach, it was also tested against T-cell acute lymphoblastic leukemia (T-ALL), another high-risk leukemia. Similarly, bortezomib increased ULBP 2/5/6 expression in T-ALL cell lines, Jurkat and MOLT-4 and improved the cytotoxicity of γδ T cells against each line. Collectively, these results show that bortezomib enhances γδ T-cell-mediated killing of both AML and T-ALL cells in part through increased NKG2D ligand-receptor interaction. Furthermore, proof-of-concept for the combination of ex vivo-expanded γδ T cells with stress ligand-inducing drugs as a therapeutic platform for high-risk leukemias is demonstrated.

Hyperleukocytosis in infant acute leukemia: a role for manual exchange transfusion for leukoreduction.

BACKGROUND: Hyperleukocytosis is a serious, life-threatening complication of pediatric acute leukemia that can cause neurologic injury, pulmonary leukostasis, metabolic derangements, and coagulopathy. Acute leukemia has the highest risk of mortality and morbidity at presentation when associated with hyperleukocytosis. Infant leukemia presents unique challenges and treatment considerations due to the disease itself and size and overall health of the patient. While medical management of hyperleukocytosis in older patients with acute leukemia has been described, including cytoreductive procedures with automated leukapheresis (AL) or manual whole blood (WB) exchange transfusion, very little data exist for standardized management of hyperleukocytosis in infant leukemia patients. CASE REPORTS: We describe four cases of infant acute leukemia presenting with hyperleukocytosis and leukostasis who each received manual WB exchange transfusions in conjunction with induction chemotherapy and review the existing literature on the use of procedural leukoreduction in infants with hyperleukocytosis. Special attention is given to challenges and technical aspects of leukapheresis in infants: when to perform manual WB exchange versus AL, optimal vascular access, blood product selection, exchange rates, and the monitoring for complications. Using published cases, we outline benefits versus risks of manual WB exchange and AL in infants less than 10 kg. CONCLUSION: If providers perform procedural leukoreduction, the literature and our experience demonstrate manual WB exchange transfusion is favored over AL in infants less than 10 kg because of technical and complication risks associated with AL. Additional studies are needed to understand the impact of cytoreduction on long-term outcomes.

Acquired Hypofibrinogenemia Before Asparaginase Exposure During Induction Therapy for Pediatric Acute Lymphoblastic Leukemia: A Report of 2 Cases and Review of the Literature.

Coagulopathy in pediatric leukemia patients is typically associated with acute promyelocytic leukemia or after asparaginase use in acute lymphoblastic leukemia. Rarely seen in acute lymphoblastic leukemia, we report 2 patients who presented with normal coagulation markers, but subsequently developed severe hypofibrinogenemia and bleeding in induction before administration of asparaginase. In both cases, cryoprecipitate was administered as initial treatment for bleeding associated with the hypofibrinogenemia. One patient was refractory to cryoprecipitate replacement and required treatment with human fibrinogen concentrate due to the persistence of hypofibrinogenemia with significant bleeding. The hypofibrinogenemia was transient in both cases and resolved within a few weeks.

Early T-Cell Precursor Acute Lymphoblastic Leukemia in an Infant With an NRAS Q61R Mutation and Clinical Features of Juvenile Myelomonocytic Leukemia.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a subtype of T-acute lymphoblastic leukemia (T-ALL) arising from a primitive precursor. We present a unique case of an infant with ETP-ALL with a missense NRAS mutation in codon 61 (c.182A>G, p.Q61R). The patient also had a minor population of non-ETP T-ALL blasts and clinical features typically associated with juvenile myelomonocytic leukemia (JMML), namely, absolute monocytosis, splenomegaly, and elevated hemoglobin F. The treatment was initiated with chemotherapy, followed by cord blood transplantation. The patient achieved remission, but unfortunately died from transplant-related complications. This case highlights an NRAS mutation in ETP-ALL with JMML-like phenotype.

Outcomes of pediatric low-grade gliomas treated with radiation therapy: a single-institution study.

Radiation therapy is often considered the treatment of choice for low-grade gliomas. However, given the long-term effects of radiation on the developing brain, the appropriate use of radiation therapy in pediatric patients remains controversial. The purpose of this study was to evaluate progression-free survival (PFS) of pediatric low-grade glioma patients treated with radiation therapy. Data were obtained through a retrospective chart review of patients treated between 1991 and 2008 from a single tertiary care center in the midwest. The study population consisted of 17 patients, of whom 8 (47%) had tumor recurrence after radiation therapy. The median follow-up time was 8.2 years, with a range of 2.3 to 17.2 years. The median age at diagnosis was 5.4 years, and the median age at radiation therapy was 9.4 years. The 3- and the 10-year PFS were 69%± 11.7% and 46%± 13.3%, respectively. A significant difference in PFS was seen when comparing brainstem tumors with hypothalamic/optic pathway tumors (P=0.019). Differences in PFS based on the age at diagnosis, the extent of initial surgery, and indication for radiation therapy were not significant. A larger multicenter study is needed to better assess PFS in these patients.

Development of a microfluidic cell transfection device into gene-edited CAR T cell manufacturing workflow.

Genetic reprogramming of immune cells to recognize and target tumor cells offers a possibility of long-term cure. Cell therapies, however, lack simple and affordable manufacturing workflows, especially to genetically edit immune cells to more effectively target cancer cells and avoid immune suppression mechanisms. Microfluidics is a pathway to improve the manufacturing precision of gene modified cells. However, to date, it remains to be demonstrated that microfluidic treatment preserves the functionality of T cell products in a complete workflow. In this study, we used microfluidics to perform CRISPR/Cas9 gene editing of CD5, a negative T-cell regulator, followed by the insertion of a chimeric antigen receptor (CAR) transgene via lentiviral vector transduction to generate CAR T cells targeted against the B cell antigen CD19. As part of the workflow, we have optimized a microfluidic device that relies on convective volume exchange between cells and surrounding fluid to deliver guide RNA and Cas9 ribonucleoprotein to primary T cells. We comprehensively tested critical design features of the device to improve the gene-edited product yield. By combining high-speed video and cell mechanics measurements using the atomic force microscope, we validate a model that relates the device design features to cell properties. Our findings showed enhanced performance was obtained by focusing the cells to counteract the flow resistance caused by the ridge constrictions, providing a ridge layout that allows sufficient cycles of compression and time for volume recovery, and including a gutter to clear aggregates that could reduce cell viability. The optimized device was used in a workflow to generate CD5-knockout CD19 CAR T cells. The microfluidics approach resulted in >60% CD5 editing efficiency, ≥80% cell viability, similar memory phenotype composition as unprocessed cells, and superior cell growth. The microfluidics workflow yielded 4-fold increase of edited T cells compared to an electroporation workflow post-expansion. The transduced CAR T cells showed similar transduction efficiency and cytotoxicity against CD19-positive leukemia cells. Moreover, patient-derived T cells showed the ability to be similarly edited, though their distinct biomechanics resulted in slightly lower outcomes. Microfluidics-based manufacturing is a promising path towards more productive clinical manufacturing of gene edited CAR T cells.

Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers.

Adoptive cell therapy (ACT) utilizing γδ T cells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific T cell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ T cells with CAR or secreted bispecific T cell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ T cells. Using a CD19-specific CAR, approximately 60% of γδ T cells are modified after mRNA electroporation and these cells show potent anticancer activity in vitro and in vivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ T cell cytotoxicity, both in vitro and in vivo, and promotes killing of target cells by modified and unmodified γδ T cells. Taken together, we show that transient transfection of γδ T cells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.

Pediatric T-cell acute lymphoblastic leukemia blast signature and MRD associated immune environment changes defined by single cell transcriptomics analysis.

Different driver mutations and/or chromosomal aberrations and dysregulated signaling interactions between leukemia cells and the immune microenvironment have been implicated in the development of T-cell acute lymphoblastic leukemia (T-ALL). To better understand changes in the bone marrow microenvironment and signaling pathways in pediatric T-ALL, bone marrows collected at diagnosis (Dx) and end of induction therapy (EOI) from 11 patients at a single center were profiled by single cell transcriptomics (10 Dx, 5 paired EOI, 1 relapse). T-ALL blasts were identified by comparison with healthy bone marrow cells. T-ALL blast-associated gene signature included SOX4, STMN1, JUN, HES4, CDK6, ARMH1 among the most significantly overexpressed genes, some of which are associated with poor prognosis in children with T-ALL. Transcriptome profiles of the blast cells exhibited significant inter-patient heterogeneity. Post induction therapy expression profiles of the immune cells revealed significant changes. Residual blast cells in MRD+ EOI samples exhibited significant upregulation (P < 0.01) of PD-1 and RhoGDI signaling pathways. Differences in cellular communication were noted in the presence of residual disease in T cell and hematopoietic stem cell compartments in the bone marrow. Together, these studies generate new insights and expand our understanding of the bone marrow landscape in pediatric T-ALL.

Single-cell RNA sequencing distinctly characterizes the wide heterogeneity in pediatric mixed phenotype acute leukemia.

BACKGROUND: Mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia characterized by blast cells with myeloid and lymphoid lineage features, is difficult to diagnose and treat. A better characterization of MPAL is essential to understand the subtype heterogeneity and how it compares with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on pediatric MPAL bone marrow (BM) samples to develop a granular map of the MPAL blasts and microenvironment landscape. METHODS: We analyzed over 40,000 cells from nine pediatric MPAL BM samples to generate a single-cell transcriptomic landscape of B/myeloid (B/My) and T/myeloid (T/My) MPAL. Cells were clustered using unsupervised single-cell methods, and malignant blast and immune clusters were annotated. Differential expression analysis was performed to identify B/My and T/My MPAL blast-specific signatures by comparing transcriptome profiles of MPAL with normal BM, AML, and ALL. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. RESULTS: B/My and T/My MPAL blasts displayed distinct blast signatures. Transcriptomic analysis revealed that B/My MPAL profile overlaps with B-ALL and AML samples. Similarly, T/My MPAL exhibited overlap with T-ALL and AML samples. Genes overexpressed in both MPAL subtypes' blast cells compared to AML, ALL, and healthy BM included MAP2K2 and CD81. Subtype-specific genes included HBEGF for B/My and PTEN for T/My. These marker sets segregated bulk RNA-seq AML, ALL, and MPAL samples based on expression profiles. Analysis comparing T/My MPAL to ETP, near-ETP, and non-ETP T-ALL, showed that T/My MPAL had greater overlap with ETP-ALL cases. Comparisons among MPAL subtypes between adult and pediatric samples showed analogous transcriptomic landscapes of corresponding subtypes. Transcriptomic differences were observed in the MPAL samples based on response to induction chemotherapy, including selective upregulation of the IL-16 pathway in relapsed samples. CONCLUSIONS: We have for the first time described the single-cell transcriptomic landscape of pediatric MPAL and demonstrated that B/My and T/My MPAL have distinct scRNAseq profiles from each other, AML, and ALL. Differences in transcriptomic profiles were seen based on response to therapy, but larger studies will be needed to validate these findings.

Single-cell analysis reveals altered tumor microenvironments of relapse- and remission-associated pediatric acute myeloid leukemia.

Acute myeloid leukemia (AML) microenvironment exhibits cellular and molecular differences among various subtypes. Here, we utilize single-cell RNA sequencing (scRNA-seq) to analyze pediatric AML bone marrow (BM) samples from diagnosis (Dx), end of induction (EOI), and relapse timepoints. Analysis of Dx, EOI scRNA-seq, and TARGET AML RNA-seq datasets reveals an AML blasts-associated 7-gene signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH), which we validate on independent datasets. The analysis reveals distinct clusters of Dx relapse- and continuous complete remission (CCR)-associated AML-blasts with differential expression of genes associated with survival. At Dx, relapse-associated samples have more exhausted T cells while CCR-associated samples have more inflammatory M1 macrophages. Post-therapy EOI residual blasts overexpress fatty acid oxidation, tumor growth, and stemness genes. Also, a post-therapy T-cell cluster associated with relapse samples exhibits downregulation of MHC Class I and T-cell regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse- and CCR-associated samples to provide insights into the BM microenvironment landscape.

Allogeneic gamma delta T cells as adoptive cellular therapy for hematologic malignancies.

Cancer immunotherapy, especially T-cell driven targeting, has significantly evolved and improved over the past decade, paving the way to treat previously refractory cancers. Hematologic malignancies, given their direct tumor accessibility and less immunosuppressive microenvironment compared to solid tumors, are better suited to be targeted by cellular immunotherapies. Gamma delta (γδ) T cells, with their unique attributes spanning the entirety of the immune system, make a tantalizing therapeutic platform for cancer immunotherapy. Their inherent anti-tumor properties, ability to act like antigen-presenting cells, and the advantage of having no major histocompatibility complex (MHC) restrictions, allow for greater flexibility in their utility to target tumors, compared to their αß T cell counterpart. Their MHC-independent anti-tumor activity, coupled with their ability to be easily expanded from peripheral blood, enhance their potential to be used as an allogeneic product. In this review, the potential of utilizing γδ T cells to target hematologic malignancies is described, with a specific focus on their applicability as an allogeneic adoptive cellular therapy product.

Obesity-induced galectin-9 is a therapeutic target in B-cell acute lymphoblastic leukemia.

The incidence of obesity is rising with greater than 40% of the world's population expected to be overweight or suffering from obesity by 2030. This is alarming because obesity increases mortality rates in patients with various cancer subtypes including leukemia. The survival differences between lean patients and patients with obesity are largely attributed to altered drug pharmacokinetics in patients receiving chemotherapy; whereas, the direct impact of an adipocyte-enriched microenvironment on cancer cells is rarely considered. Here we show that the adipocyte secretome upregulates the surface expression of Galectin-9 (GAL-9) on human B-acute lymphoblastic leukemia cells (B-ALL) which promotes chemoresistance. Antibody-mediated targeting of GAL-9 on B-ALL cells induces DNA damage, alters cell cycle progression, and promotes apoptosis in vitro and significantly extends the survival of obese but not lean mice with aggressive B-ALL. Our studies reveal that adipocyte-mediated upregulation of GAL-9 on B-ALL cells can be targeted with antibody-based therapies to overcome obesity-induced chemoresistance.

Mixed phenotype acute leukemia in a child associated with a NUP98-NSD1 fusion and NRAS p.Gly61Arg mutation.

BACKGROUND: Mixed phenotype acute leukemia (MPAL) is a rare subset of acute leukemia in the pediatric population associated with genetic alterations seen in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). CASE: We describe a patient with MPAL with a NUP98 (nucleoporin 98)-NSD1 gene fusion (nuclear receptor binding SET domain protein1) and NRAS (neuroblastoma RAS viral oncogene homolog mutation) p.Gly61Arg mutation who was treated with upfront AML-based chemotherapy, received hematopoietic stem cell transplant (HSCT), but unfortunately died from relapsed disease. CONCLUSION: This case highlights the challenges faced in choosing treatment options in MPAL patients with complex genomics, with predominant myeloid features.

Interleukin-37 improves T-cell-mediated immunity and chimeric antigen receptor T-cell therapy in aged backgrounds.

Aging-associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world's population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging-associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin-37 (IL-37) is a potent anti-inflammatory cytokine, and we present data demonstrating that IL-37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin-37 (IL-37) in aged mice reduces or prevents aging-associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL-37 expression decreases the surface expression of programmed cell death protein 1 (PD-1) and augments cytokine production from aged T-cells. Improved T-cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4+ T-cells and Lat in CD8+ T-cells when aged mice were treated with recombinant IL-37 (rIL-37) but not control immunoglobin (Control Ig). Importantly, IL-37-mediated rejuvenation of aged endogenous T-cells was also observed in aged chimeric antigen receptor (CAR) T-cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL-37 in boosting the function of aged T-cells and highlight its therapeutic potential to overcome aging-associated immunosenescence.

The fantastic journey of a bullet: out with a snare.

Bullet embolus is a rare complication of penetrating missile trauma. Removal of the bullet previously required surgery. We report the case of a 14-year-old with an hepatic vein bullet embolus following a gunshot wound to the left buttock. A transjugular approach was used to extract the bullet percutaneously with an Amplatzer gooseneck snare.

Significance of minimal residual disease in pediatric mixed phenotype acute leukemia: a multicenter cohort study.

The rarity of mixed phenotype acute leukemia (MPAL) has precluded adequate data to incorporate minimal residual disease (MRD) monitoring into therapy. Fluidity in MPAL classification systems further complicates understanding its biology and outcomes; this includes uncertainty surrounding the impact of shifting diagnostic requirements even between iterations of the World Health Organization (WHO) classification. Our primary objective was to address these knowledge gaps. To do so, we analyzed clinicopathologic features, therapy, MRD, and survival in a centrally-reviewed, multicenter cohort of MPAL uniformly diagnosed by the WHO classification and treated with acute lymphoblastic leukemia (ALL) regimens. ALL induction therapy achieved an EOI MRD negative (<0.01%) remission in most patients (70%). EOI MRD positivity was predictive of 5-year EFS (HR = 6.00, p < 0.001) and OS (HR = 9.57, p = 0.003). Patients who cleared MRD by EOC had worse survival compared with those EOI MRD negative. In contrast to adults with MPAL, ALL therapy without transplantation was adequate to treat most pediatric patients. Earlier MRD clearance was associated with better treatment success and survival. Prospective trials are now necessary to validate and refine MRD thresholds within the pediatric MPAL population and to identify salvage strategies for those with poor predicted survival.