CAR T cell Toxicities: Bedside to Bench - How Novel Toxicities Inform Laboratory Investigations.

Multiple chimeric antigen receptor (CAR) T cell therapies are FDA approved, and several are under development. While effective for some cancers, toxicities remain a limitation. The most common toxicities, i.e. cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS), are well described. With increasing utilization, providers worldwide are reporting on other emergent, and often complicated toxicities. Given the evolving toxicity profiles and urgent need to catalogue these emerging and emergent CAR T toxicities and describe management approaches, the American Society of Hematology Subcommittee on Emerging Gene and Cell Therapies organized the first Scientific Workshop on CAR T cell toxicities during the annual society meeting. The workshop functioned to 1) aggregate reports of CAR T emergent toxicities, including movement disorders after BCMA CAR T, coagulation abnormalities, and prolonged cytopenias; 2) disseminate bedside to bench efforts elucidating pathophysiological mechanisms of CAR-T toxicities, including the intestinal microbiota and systemic immune dysregulation; and 3) highlight gaps in the availability of clinical tests such as cytokine measurements, which could be utilized to expand our knowledge around the monitoring of toxicities. Key themes emerged. First, while clinical manifestations may develop before the pathophysiologic mechanisms are understood, these must be studied to aid in the detection and prevention of such toxicities. Second, systemic immune dysregulation appears central to these emergent toxicities and research is needed to elucidate links between tumor, CAR T, and microbiota. Finally, there was consensus around an urgency to create a repository to capture emergent CAR-T toxicities and the real-world management.

mRNA-based therapeutic strategies for cancer treatment.

In the rapidly evolving landscape of medical research, the emergence of RNA-based therapeutics is paradigm shifting. It is mainly driven by the molecular adaptability and capacity to provide precision in targeting. The coronavirus disease 2019 pandemic crisis underscored the effectiveness of the mRNA therapeutic development platform and brought it to the forefront of RNA-based interventions. These RNA-based therapeutic approaches can reshape gene expression, manipulate cellular functions, and correct the aberrant molecular processes underlying various diseases. The new technologies hold the potential to engineer and deliver tailored therapeutic agents to tackle genetic disorders, cancers, and infectious diseases in a highly personalized and precisely tuned manner. The review discusses the most recent advancements in the field of mRNA therapeutics for cancer treatment, with a focus on the features of the most utilized RNA-based therapeutic interventions, current pre-clinical and clinical developments, and the remaining challenges in delivery strategies, effectiveness, and safety considerations.

Translation efficiency driven by CNOT3 subunit of the CCR4-NOT complex promotes leukemogenesis.

Protein synthesis is frequently deregulated during tumorigenesis. However, the precise contexts of selective translational control and the regulators of such mechanisms in cancer is poorly understood. Here, we uncovered CNOT3, a subunit of the CCR4-NOT complex, as an essential modulator of translation in myeloid leukemia. Elevated CNOT3 expression correlates with unfavorable outcomes in patients with acute myeloid leukemia (AML). CNOT3 depletion induces differentiation and apoptosis and delayed leukemogenesis. Transcriptomic and proteomic profiling uncovers c-MYC as a critical downstream target which is translationally regulated by CNOT3. Global analysis of mRNA features demonstrates that CNOT3 selectively influences expression of target genes in a codon usage dependent manner. Furthermore, CNOT3 associates with the protein network largely consisting of ribosomal proteins and translation elongation factors in leukemia cells. Overall, our work elicits the direct requirement for translation efficiency in tumorigenesis and propose targeting the post-transcriptional circuitry via CNOT3 as a therapeutic vulnerability in AML.

The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of ß-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.

Corrigendum: Protein arginine methyltransferase 1 is a therapeutic vulnerability in multiple myeloma.

[This corrects the article DOI: 10.3389/fimmu.2023.1239614.].

Pharmacologic targeting of the p62 ZZ domain enhances both anti-tumor and bone-anabolic effects of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades Narginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib by increasing: 1) killing of human MM cells by stimulating both bortezomib mediated apoptosis and necroptosis, a process regulated by p62; and 2) preservation of bone mass by stimulating osteoblasts differentiation and inhibiting osteoclastic bone destruction. Co-administration of bortezomib and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, coadministration of bortezomib and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti-MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.

Protein arginine methyltransferase 1 is a therapeutic vulnerability in multiple myeloma.

Multiple myeloma (MM) is a devastating plasma cell malignancy characterized by the expansion of aberrant monoclonal plasma cells in the bone marrow, leading to severe clinical manifestations and poor prognosis, particularly in relapsed/refractory cases. Identifying novel therapeutic targets is crucial to improve treatment outcomes in these patients. In this study, we investigated the role of the protein arginine methyltransferase 1 (PRMT1) in MM pathogenesis and explored its potential as a therapeutic target. We observed that PRMT1, responsible for most asymmetric di-methylation in cells, exhibited the highest expression among PRMT family members in MM cell lines and primary MM cells. Importantly, PRMT1 expression was significantly elevated in relapsed/refractory patients compared to newly diagnosed patients. High expression of PRMT1 expression was strongly associated with poor prognosis. We found that genetic or enzymatic inhibition of PRMT1 impaired MM cell growth, induced cell cycle arrest, and triggered cell death. Treatment with MS023, a potent PRMT type I inhibitor, demonstrated a robust inhibitory effect on the viability of primary cells isolated from newly diagnosed and proteasome inhibitor-relapsed/refractory patients in a dose-dependent manner. Suppression of PRMT1 downregulated genes related to cell division and upregulated genes associated with apoptosis pathway. We also found that genes related to immune response and lymphocyte activation were significantly upregulated in PRMT1-suppressed cells. Notably, the activation status of T cells was strikingly enhanced upon co-culturing with PRMT1-KO MM cells. In vivo studies using a xenograft model revealed that targeting PRMT1 by either CRISPR/Cas9-mediated knockout or MS023 treatment significantly attenuated MM tumor growth and prolonged the survival of tumor-bearing mice. Histological analysis further confirmed increased apoptotic cell death in MS023-treated tumors. Collectively, our findings establish PRMT1 as an indispensable and novel therapeutic vulnerability in MM. The elevated expression of PRMT1 in relapsed/refractory patients underscores its potential as a target for overcoming treatment resistance. Moreover, our results highlight the efficacy of MS023 as a promising therapeutic agent against MM, offering new avenues for therapeutic approaches in relapsed/refractory MM.

A target discovery pipeline identified ILT3 as a target for immunotherapy of multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.

Thrombotic Events Are Unusual Toxicities of Chimeric Antigen Receptor T-Cell Therapies.

Chimeric antigen receptor (CAR) T-cell therapy has greatly transformed the treatment and prognosis of B-cell hematological malignancies. As CAR T-cell therapy continues to be more readily adopted and indications increase, the field's recognition of emerging toxicities will continue to grow. Among the adverse events associated with CAR T-cell therapy, cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity (ICANS) are the most common toxicities, while thrombotic events represent an under-reported, life-endangering complication. To determine thrombosis incidence post CAR T-cell therapy, we performed a multi-center, retrospective study on CAR T-cell therapy adult patients (N = 140) from Indiana University Simon Cancer Center and the University of North Carolina Medical Center treated from 2017 to 2022 for relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL, N = 3), diffuse large B-cell lymphoma (DLBCL, N = 92), follicular lymphoma (FL, N = 9), mantle cell lymphoma (MCL, N = 2), and multiple myeloma (MM, N = 34). We report 10 (7.14%) thrombotic events related to CAR T-cell therapy (DLBCL: N = 8, FL: N = 1, MM: N = 1) including 9 primary venous events and 1 arterial event that occurred with median time of 23.5 days post CAR T-cell infusion. In search of parameters associated with such events, we performed multivariate analyses of coagulation parameters (i.e., PT, PTT, and D-Dimer), scoring for adverse events (Padua Score and ISTH DIC Score) and grading for CAR T-cell toxicity severity (CRS grade and ICANS grade) and found that D-Dimer peak elevation and ICANS grade were significantly associated with post-CAR T-cell infusion thrombosis. While the pathophysiology of CAR T-cell associated coagulopathy remains unknown, our study serves to develop awareness of these emerging and unusual complications.

How I treat unique and difficult-to-manage cases of CAR T-cell therapy-associated neurotoxicity.

With growing indications for chimeric antigen receptor (CAR) T-cell therapy, toxicity profiles are evolving. There is an urgent and unmet need of approaches to optimally manage emerging adverse events that extend beyond the standard paradigm of cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome (ICANS). Although management guidelines exist for ICANS, there is little guidance on how to approach patients with neurologic comorbidities, and how to manage rare neurotoxicity presentations, such as CAR T-cell therapy-related cerebral edema, severe motor complications or late-onset neurotoxicity. In this study, we present 3 scenarios of patients treated with CAR T cells who develop unique types of neurotoxicity, and we describe an approach for the evaluation and management based on experience because objective data are limited. The goal of this study is to develop an awareness of emerging and unusual complications, discuss treatment approaches, and help institutions and health care providers establish frameworks to navigate how to best address unusual neurotoxicities to ultimately improve patient outcomes.

Stepwise activities of mSWI/SNF family chromatin remodeling complexes direct T cell activation and exhaustion.

Highly coordinated changes in gene expression underlie T cell activation and exhaustion. However, the mechanisms by which such programs are regulated and how these may be targeted for therapeutic benefit remain poorly understood. Here, we comprehensively profile the genomic occupancy of mSWI/SNF chromatin remodeling complexes throughout acute and chronic T cell stimulation, finding that stepwise changes in localization over transcription factor binding sites direct site-specific chromatin accessibility and gene activation leading to distinct phenotypes. Notably, perturbation of mSWI/SNF complexes using genetic and clinically relevant chemical strategies enhances the persistence of T cells with attenuated exhaustion hallmarks and increased memory features in vitro and in vivo. Finally, pharmacologic mSWI/SNF inhibition improves CAR-T expansion and results in improved anti-tumor control in vivo. These findings reveal the central role of mSWI/SNF complexes in the coordination of T cell activation and exhaustion and nominate small-molecule-based strategies for the improvement of current immunotherapy protocols.

Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway.

Loss-of-function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of patients with acute myeloid leukemia (AML) with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early preleukemic event, which - when combined with other genetic lesions - result in full-blown leukemia. Here, we show that loss of Dnmt3a in hematopoietic stem and progenitor cells (HSC/Ps) results in myeloproliferation, which is associated with hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway. PI3Kα/ß or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/ß inhibitor treatment. In vivo RNA-Seq analysis on drug-treated Dnmt3a-/- HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment, and extracellular matrix compared with controls. Remarkably, drug-treated leukemic mice showed a reversal in the enhanced fetal liver HSC-like gene signature observed in vehicle-treated Dnmt3a-/- LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions, including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/ß inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a potentially new target for treating DNMT3A mutation-driven myeloid malignancies.

Clinicogenomic Landscape of Metastatic Thymic Epithelial Tumors.

BACKGROUND: Despite favorable clinical outcomes, a subset of patients with thymic epithelial tumors (TETs) develop metastasis. The Cancer Genome Atlas (TCGA) provides genomic data on primary TETs (pTETs). This study assessed the molecular alterations and uncovered targetable pathways in metastatic TETs (mTETs). METHODS: From 2015 to 2020, 49 patients with stage IV TETs underwent Clinical Laboratory Improvement Amendments-based sequencing using whole-exome sequencing (n = 33), panel-based testing (n = 12), and/or liquid biopsy (n = 24). Specimens were obtained from a metastatic organ (n = 36) or relapsed primary mediastinal mass (n = 10), whereas four patients underwent a liquid biopsy only. Data on pTETs were derived from the TCGA. RESULTS: Compared with the pTET data set, patients with mTETs were younger (54 years v 60.5 years, P = .009) and had more aggressive histologies, with the most common tumor type being thymic carcinoma (n = 22, 40.7%) and B3 thymoma (n = 15, 27.8%). GTF2I was the most altered gene in primary thymomas (48.80%, n = 60). In metastatic thymoma and thymic carcinoma, TP53 was the most common genetic alteration (31% and 36%, respectively). In mTETs, the genomic alteration occurred in the TP53/CDK, EGFR/RAS, and PI3K/mTOR pathways. Biopsies obtained from distant metastasis were more commonly found to contain targetable mutations. There was an overlap of 61% (22 of 36) between tissue and liquid biopsy genomic alterations. CONCLUSION: Clinically actionable genomic alterations are frequently observed in mTETs, indicating a value of repeat biopsy (preferably from a metastatic site of TETs for sequencing at the time of recurrence (TCGA data).

Topical Steroids and Glaucoma Filtration Surgery Outcomes: An In Vivo Confocal Study of the Conjunctiva.

(1) Background: The purpose of this study is to investigate the effects of topical steroids on conjunctiva in patients undergoing filtration surgery (FS) for glaucoma by using confocal microscopy (CM); (2) Methods: One hundred and four glaucomatous patients were randomized to fluorometholone or lubricants four weeks before FS. CM was performed before treatments and pre-operatively. Dendritic and goblet cell densities (DCD, GCD), stromal meshwork reflectivity (SMR), vascular tortuosity (VT), and intra-ocular pressure (IOP) were the main outcomes. By evaluating treatments and outcomes (12-month success/failure) as categorical variables, patients were grouped into Group 1, 2, 3, or 4 (success/failure with fluorometholone, or lubricants); (3) Results: Twelve-month IOP was reduced in Groups 1 and 3 (p < 0.001). After treatments, DCD and SMR were reduced in Groups 1 and 2 (p < 0.01), and 1 and 3 (p < 0.05), respectively. Pre-operative DCD was lower in the steroid compared to lubricant group (p < 0.001), whereas SMR was lower in successful (1 and 3) compared to failed groups (2 and 4) (p = 0.004). There were no significant differences between the fluorometholone and lubricant groups for success percentages. The number of bleb management procedures and IOP lowering medications were lower in Group 1 compared to Groups 2−4 (p < 0.05); (4) Conclusions: Topical steroids mitigate conjunctival inflammation and lower the stromal density in patients undergoing FS. These modifications lead to less intensive post-operative management.

A case report of the metagenomics next-generation sequencing for early detection of central nervous system mucormycosis with successful rescue in patient with recurrent chronic lymphocytic leukemia.

Background: Central nervous system (CNS) mucormycosis is insidious and difficult to diagnose. It progresses rapidly and causes high mortality. Rare cases have been reported during ibrutinib use, which have poor prognosis. Through this case, we share the experience of successful diagnosis and treatment. We also emphasize the importance of focusing on high-risk groups, early diagnosis and prompt management. Case Description: In this case, a 52-year-old patient was diagnosed with chronic lymphocytic leukemia (CLL) for more than 5 years. He was in remission after rituximab plus fludarabine and cyclophosphamide (RFC) regimen, and relapsed in the fourth year. During the ibrutinib monotherapy, the patient presented with sudden headache. Cranial imaging examination revealed a definite right occipitoparietal lobe mass with extensive edema. A rapid diagnosis of mucormycosis infection was made using metagenomic next-generation sequencing (mNGS). The patient at that time didn't have neutropenia, but he had hypogammaglobulinemia. The infection was treated with amphotericin B cholesteryl sulfate complex, posaconazole, and interventional surgery, and the treatment was successful. At the same time, we considered the control of disease progression in this relapsed patient with, as well as to the drug interaction with posaconazole. We chose the next generation Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib as the treatment, whose safety has been identified. As of the submission date, the patient has been followed up for nearly 1 year, and his disease is stable. Conclusions: When new clinical problems arise in recurrent CLL patients, it is important to identify multiple factors, especially the insidious fungal infections. In particular, the immunocompromised patients should be concerned. CNS mucormycosis is extremely deadly, the early diagnosis will improve the prognosis. In clinical practice, the gold standard diagnosis of mucormycosis is difficult to obtain through pathology. In this case, mNGS was applied to quickly diagnose mucormycosis, enabling earlier treatment and ameliorating the prognosis. Thus, it will help us to early detect this group of people who may be potentially infected. Current guidelines do not recommend the prophylactic use of antifungal agents in treated CLL patients. However, in patients with prior severe infection or hypogammaglobulinemia, intravenous immunoglobulin is recommended to reduce the associated infection rate.

Novel Immune-Based treatments for Diffuse Large B-Cell Lymphoma: The Post-CAR T Cell Era.

Prognosis for patients with refractory/relapsed (R/R) diffuse large B-cell lymphoma (DLBCL) is poor. Immune-based therapeutic treatments such as CD19 Chimeric Antigen Receptor (CAR) T cell therapies have dramatically changed the treatment landscape for R/R DLBCL leading to durable remissions in ~ 50% of patients. However, there remains an unmet need for developing novel therapies to improve clinical outcomes of patients not responding or relapsing after CAR T cell therapies. Lack of suitable immunotherapeutic targets and disease heterogeneity represent the foremost challenges in this emerging field. In this review, we discuss the recently approved and emerging novel immunotherapies for patients with R/R DLBCL in the post-CAR T era and the cell surface targets currently used.

Immune-Based Therapeutic Interventions for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive, clonally heterogeneous, myeloid malignancy, with a 5-year overall survival of approximately 27%. It constitutes the most common acute leukemia in adults, with an incidence of 3-5 cases per 100,000 in the United States. Despite great advances in understanding the molecular mechanisms underpinning leukemogenesis, the past several decades had seen little change to the backbone of therapy, comprised of an anthracycline-based induction regimen for those who are fit enough to receive it, followed by risk-stratified post-remission therapy with consolidation cytarabine or allogeneic stem cell transplantation (allo-SCT). Allo-SCT is the most fundamental form of immunotherapy in which donor cytotoxic T and NK cells recognize and eradicate residual AML in the graft-versus-leukemia (GvL) effect. Building on that, several alternative or synergistic approaches to exploit both self and foreign immunity against AML have been developed. Checkpoint inhibitors, for example, CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors block proteins found on T cells or cancer cells that stop the immune system from attacking the cancer cells. They have been used with limited success in both the AML relapsed/refractory (R/R) and post SCT settings. AML tumor mutational burden is low compared to solid tumors and thus, it is less likely to generate neoantigens and respond to antibody-mediated checkpoint blockade that has shown unprecedented results in solid tumors. Therefore, alternative therapeutic strategies that work independently of the T cell receptor (TCR) specificity have been developed. They include bispecific antibodies, which recruit T cells through CD3 engagement, and in AML have shown an overall response rate ranging between 14 and 30% in early phase trials. Chimeric Antigen Receptor (CAR) T cell therapy is a type of treatment in which T cells are genetically engineered to produce a recombinant receptor that redirects the specificity and function of T lymphocytes. However, lack of cell surface targets exclusively expressed on AML cells including Leukemic Stem Cells (LSCs) combined with clonal heterogeneity represents the biggest challenge in developing CAR therapy for AML. Antibody-Drug Conjugates (ADC) constitute the only FDA-approved immunotherapy to treat AML with Gemtuzumab Ozogamicin, a CD33-specific ADC used in CEBPα-mutated AML. The identification of additional cell surface targets is critical for the development of other ADC's potentially useful in the induction and maintenance regimens, given the ease at which these reagents can be generated and managed. Here, we will review those immune-based therapeutic interventions and highlight active areas of research investigations toward fulfillment of the great promise of immunotherapy to AML.

Targets for chimeric antigen receptor T-cell therapy of acute myeloid leukemia.

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy associated with high mortality rates (less than 30% 5-year survival). Despite advances in our understanding of the molecular mechanisms underpinning leukemogenesis, standard-of-care therapeutic approaches have not changed over the last couple of decades. Chimeric Antigen Receptor (CAR) T-cell therapy targeting CD19 has shown remarkable clinical outcomes for patients with acute lymphoblastic leukemia (ALL) and is now an FDA-approved therapy. Targeting of myeloid malignancies that are CD19-negative with this promising technology remains challenging largely due to lack of alternate target antigens, complex clonal heterogeneity, and the increased recognition of an immunosuppressive bone marrow. We carefully reviewed a comprehensive list of AML targets currently being used in both proof-of-concept pre-clinical and experimental clinical settings. We analyzed the expression profile of these molecules in leukemic as well normal tissues using reliable protein databases and data reported in the literature and we provide an updated overview of the current clinical trials with CAR T-cells in AML. Our study represents a state-of-art review of the field and serves as a potential guide for selecting known AML-associated targets for adoptive cellular therapies.