Proapoptotic and immunomodulatory effects of SYK inhibitor entospletinib in combination with obinutuzumab in patients with chronic lymphocytic leukaemia.

Spleen tyrosine kinase (SYK) is indispensable in B-cell receptor signalling. SYK inhibitor entospletinib demonstrated clinical efficacy in patients with chronic lymphocytic leukaemia (CLL). However, pharmacodynamic effects of SYK inhibition in CLL cells and immunomodulatory effects of B-cell receptor-signalling inhibitors in patients with CLL are poorly understood. We conducted a phase 2 trial of entospletinib in combination with obinutuzumab, an anti-CD20 antibody, in 17 patients with relapsed/refractory CLL. Pharmacodynamic analysis demonstrated that treatment with entospletinib led to rapid downmodulation of pSTAT3 and the anti-apoptotic protein MCL1 in CLL cells. Meanwhile, 6 months of combination therapy was accompanied by a reduction in interferon-γ secretion in CD4+ T-cells and a reversal of exhausted phenotype, as evidenced by downregulation of PD-1. Thus, SYK inhibition downmodulates MCL-1 and partially restores T-cell immunity in CLL. Trial registration number NCT03010358.

SYK inhibition thwarts the BAFF - B-cell receptor crosstalk and thereby antagonizes Mcl-1 in chronic lymphocytic leukemia.

Although small molecule inhibitors of B-cell receptor-associated kinases have revolutionized therapy in chronic lymphocytic leukemia (CLL), responses are incomplete. Pro-survival signaling emanating from the microenvironment may foster therapeutic resistance of the malignant B cells resident in the protective lymphoid niches. B-cell activating factor (BAFF) is critical to the survival of both healthy and neoplastic B cells. However, the pro-survival pathways triggered by BAFF have not been fully characterized. Here we show that BAFF elicited resistance to spontaneous and drug-induced apoptosis in stromal co-cultures, induced activation of both canonical and non-canonical NFκB signaling pathways, and triggered B-cell receptor signaling in CLL cells, independently of IGHV mutational status. SYK, a proximal kinase in the B-cell receptor signaling cascade, acted via STAT3 to bolster transcription of the anti-apoptotic protein Mcl-1, thereby contributing to apoptosis resistance in BAFF-stimulated cells. SYK inhibitor entospletinib downregulated Mcl-1, abrogating BAFF-mediated cell survival. BAFF-B-cell receptor crosstalk in neoplastic B cells was mediated by SYK interaction with TRAF2/TRAF3 complex. Thus, SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and B-cell receptor, thereby disrupting the pro-survival microenvironment signaling in chronic lymphocytic leukemia.

T Cell-intrinsic Immunomodulatory Effects of TAK-981 (Subasumstat), a SUMO-activating Enzyme Inhibitor, in Chronic Lymphocytic Leukemia.

Novel targeted agents used in therapy of lymphoid malignancies are recognized to have complex immune-mediated effects. Sumoylation, a posttranslational modification of target proteins by small ubiquitin-like modifiers (SUMO), regulates a variety of cellular processes indispensable in immune cell activation. Despite this, the role of sumoylation in T-cell biology in context of cancer is not known. TAK-981 (subasumstat) is a small-molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein. Using T cells derived from patients with chronic lymphocytic leukemia (CLL), we demonstrate that targeting SAE activates type I IFN response. This is accompanied by largely intact T-cell activation in response to T-cell receptor engagement, with increased expression of CD69 and CD38. Furthermore, TAK-981 decreases regulatory T cell (Treg) differentiation and enhances secretion of IFNγ by CD4+ and CD8+ T cells. These findings were recapitulated in mouse models, suggesting an evolutionarily conserved mechanism of T-cell activation regulated by SUMO modification. Relevant to the consideration of TAK-981 as an effective agent for immunotherapy in hematologic malignancies, we demonstrate that the downstream impact of TAK-981 administration is enhancement of the cytotoxic function of CD8+ T cells, thus uncovering immune implications of targeting sumoylation in lymphoid neoplasia.

Qualitative Perceptions of an Anticipated Fresh Food Prescription Program.

Objective: Food insecurity (FI) is a growing public health problem. Produce prescriptions are known to improve healthy eating and decrease FI; however, few studies have incorporated community voice prior to its implementation. In this study, we aimed to elicit perspectives of individuals at risk for FI and the potential impact of a fresh food prescription (FFRx) program. Methods: We conducted this qualitative descriptive study through an academic medical center in collaboration with community partners. We conducted focus groups involving Latinx (N = 16) and African-American (N = 8) adults in community settings. Data were interpreted using an inductive thematic analysis. Results: Three overarching themes emerged: (1) fresh food accessibility was limited by cost, household size, and transportation but enhanced by food pantries, budgeting, and education; (2) cooking behaviors were curbed by time constraints and unfamiliarity but propagated by passion, traditions, and communal practices; and (3) health and wellness deterrents included unhealthy diets driven by cultural and familial norms; however, weight loss and awareness of comorbidities were positive motivators. Participants shared their preference for local produce and cooking classes as components of a FFRx program while raising concerns about low participation due to the stigma of receiving aid. Conclusions: Our findings illuminated interest in engaging in a FFRx program and learning ways to prepare healthy foods. A program distributing fresh produce and healthy lifestyle education could close gaps identified in African-American and Latinx communities at risk for FI.

Exploring Perceptions of a Fresh Food Prescription Program during COVID-19.

This qualitative study aimed to elicit the perspectives of individuals with food insecurity (FI) who were enrolled in a Fresh Food Prescription (FFRx) delivery program through a collaboration between an academic medical center and multiple community partners in the southeastern United States. Semi-structured interviews and open-ended survey responses explored the experiences of participants enrolled in a FFRx delivery program during the COVID-19 pandemic. The interviews probed the shopping habits, food security, experience, and impact of the program on nutrition, health, and well-being; the surveys explored the perceptions of and satisfaction with the program. A coding scheme was developed inductively, and a thematic analysis was conducted on raw narrative data using Atlas.ti 8.4 to sort and manage the data. The themes included that the program promoted healthy dietary habits, improved access to high-quality foods, improved well-being, enhanced financial well-being, and alleviated logistical barriers to accessing food and cooking. Participants provided suggestions for FFRx improvement. Future studies may facilitate improved clinical-community partnerships to address FI.

Strategies and Lessons Learned from a Home Delivery Food Prescription Program for Older Adults.

Food insecurity (FI) is a growing health problem, worsening during the COVID-19 pandemic. Fresh food prescription programs (FFRx) have been shown to increase healthy eating and decrease FI, but few FFRx are community-informed, or theory based. Our FFRx was a delivery program developed to alleviate FI for older adults. It was implemented in an academic medical center and guided by the Capabilities, Opportunities, Motivations, and Behaviors and Theoretical Domains Framework. We tested impacts of the program on FI, Fruit and Vegetable (FV) intake, depression, and loneliness at six-month intervals. During the FFRx, 31 people completed surveys every six months. FI decreased by an average of 2.03 points (p = <.001) while FV intake increased from a mean of 2.8 servings per day to 2.9 servings per day (p = .53). Depression and loneliness scores stayed stable. Preliminary data from this FFRx program, a partnership between an academic medical center and community partners, had positive impacts on FI.

Neddylation and anti-tumor immunity.

Contrary to chemotherapy, novel targeted therapies are associated with diverse immunomodulatory effects. Nedd8 is a small ubiquitin-like modifier that is involved in regulation of protein degradation. Neddylation is a promising target in cancer. Pevonedistat, a small molecule inhibitor of the Nedd8-activating enzyme, demonstrates pre-clinical activity in multiple tumor types. Recent studies have revealed that neddylation is important in immunity. We and others have shown that interfering with neddylation causes downstream immunomodulatory effects potentially leading to enhanced anti-tumor immunity. Thus, Nedd8 is a promising target in immuno-oncology.

Engaging Food Insecure Families on the Weekend to Improve Nutrition and Health.

BACKGROUND: The school meals program provides food during the week, but there is limited evidence on how to address the needs of families with food insecurity (FI) on the weekend. OBJECTIVES: We conducted a prospective mixed methods pilot study to evaluate the potential effect of a community-based program that delivers free meals to children and fresh produce to their families at different sites on weekends combined with cooking classes. METHODS: We recruited 41 parent-child dyads from the neighborhood where a new delivery site opened. We assessed the change in children's fruit and vegetable intake and parental anxiety before and after the site opened. We conducted interviews with parents/guardians to understand perceptions of the program and how to more effectively address families' needs. RESULTS: The majority of parents/guardians were non-Hispanic Black (90.5%) and had FI (87.8%). We found a non-significant increase in the intake of fruit/vegetable servings per day from baseline (mean, 3.39) to follow-up (mean, 3.88; p = 0.41), but no change in parental anxiety. In interviews, we identified three major themes: 1) FI affects food quality and multiple generations care for children; 2) the program provided relief to parents/guardians, allowing them to eat healthier; and 3) the need for multigenerational programs and broader policy changes to address FI. CONCLUSIONS: Although further research is needed, a community-based program combining food delivery with cooking classes may assist families with FI. Participants reported that the program provided relief, helping them eat healthier. Multigenerational programs and broader policy changes are needed to further address FI.

NEDD8-activating enzyme inhibition induces cell cycle arrest and anaphase catastrophe in malignant T-cells.

Peripheral T-cell lymphoma (PTCL) is characterized by poor outcomes. We and others have shown that targeting the NEDD8-activating enzyme (NAE) with an investigational inhibitor pevonedistat deregulates cell cycle and mitosis in lymphoma and leukemia. Here, we report that PTCL is characterized by increased rate of chromosomal instability. NAE inhibition promotes cell cycle arrest and induces multipolar anaphases in T-cell lymphoma cell lines, resulting in apoptosis, also observed in primary malignant PTCL cells treated with pevonedistat. We identified p27Kip1 as a mediator of anaphase catastrophe in these cells. Targeting neddylation with pevonedistat may be a promising approach to treatment of PTCL.

Immunomodulatory effects of pevonedistat, a NEDD8-activating enzyme inhibitor, in chronic lymphocytic leukemia-derived T cells.

Novel targeted agents used in therapy of lymphoid malignancies, such as inhibitors of B-cell receptor-associated kinases, are recognized to have complex immune-mediated effects. NEDD8-activating enzyme (NAE) has been identified as a tractable target in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma. We and others have shown that pevonedistat (TAK-924), a small-molecule inhibitor of NAE, abrogates NF-κB signaling in malignant B cells. However, NF-κB pathway activity is indispensable in immune response, and T-cell function is altered in patients with CLL. Using T cells derived from patients with CLL, we demonstrate that although targeting NAE results in markedly differential expression of NF-κB-regulated genes and downregulation of interleukin (IL)-2 signaling during T-cell activation, T cells evade apoptosis. Meanwhile, NAE inhibition favorably modulates polarization of T cells in vitro, with decreased Treg differentiation and a shift toward TH1 phenotype, accompanied by increased interferon-γ production. These findings were recapitulated in vivo in immunocompetent mouse models. T cells exposed to pevonedistat in washout experiments, informed by its human pharmacokinetic profile, recover NAE activity, and maintain their response to T-cell receptor stimulation and cytotoxic potential. Our data shed light on the potential immune implications of targeting neddylation in CLL and lymphoid malignancies.

What a city eats: Examining the dietary preferences of families living in communities at high risk for food insecurity.

INTRODUCTION: Food insecurity (FI) is the lack of consistent access to enough food for an active and healthy life. Community-based hunger relief programs often serve as emergency food sources for families with FI. However, these programs may not provide foods that diverse populations of people prefer. We sought to evaluate the dietary patterns and preferences of families living in food-insecure neighborhoods and utilizing a community-based hunger relief program, in order to improve the utilization of local nutritional programs. METHODS: We examined the Help Our People Eat (HOPE) community-based mobile meal program. Free-listing interviews (n = 63) were conducted with English-(66%) and Spanish-speaking (34%) participants of the program. Participants were asked about FI risk, food preferences, and dietary behaviors at home. RESULTS: The majority of participants (90%) had children in the household. About 60% reported not being able to afford the type of food they enjoyed. Most participants reported using stoves for cooking (80%). Participants overwhelmingly cooked with chicken, beef, and pork. The most common side dishes included potatoes, rice, and salad. Most participants reported no interest in cooking differently or learning new recipes. CONCLUSIONS: A common theme throughout interviews was that families prefer similar meals, but may prepare them differently based on the language spoken. Food preferences consisted of a high intake of carbohydrate-rich meals, perhaps because these foods may be cheaper and easier to access. Notably, new recipes and cooking methods were not a priority for these families, possibly due to the time and effort needed to learn them.

Pharmacologic inhibition of the ubiquitin-activating enzyme induces ER stress and apoptosis in chronic lymphocytic leukemia and ibrutinib-resistant mantle cell lymphoma cells.

With the advent of proteasome inhibitors (bortezomib) and pleiotropic pathway modulators which target cereblon E3 ligase (lenalidomide), the ubiquitin-proteasome system has emerged as a tractable target in non-Hodgkin lymphoma and multiple myeloma. Here we report that TAK-243, a small molecule inhibitor of the ubiquitin-activating enzyme (UAE), induced ER stress and the unfolded protein response in primary chronic lymphocytic leukemia cells, facilitating cell death. Moreover, targeting UAE was effective in ibrutinib-resistant mantle cell lymphoma cell lines and primary cells in vitro. Thus, UAE is a promising target in lymphoid malignancies, including ibrutinib-resistant lymphomas, an area of unmet medical need.

Targeting ubiquitin-activating enzyme induces ER stress-mediated apoptosis in B-cell lymphoma cells.

Alterations in the ubiquitin proteasome system (UPS) leave malignant cells in heightened cellular stress, making them susceptible to proteasome inhibition. However, given the limited efficacy of proteasome inhibitors in non-Hodgkin lymphoma (NHL), novel approaches to target the UPS are needed. Here, we show that TAK-243, the first small-molecule inhibitor of the ubiquitin activating enzyme (UAE) to enter clinical development, disrupts all ubiquitin signaling and global protein ubiquitination in diffuse large B-cell lymphoma (DLBCL) cells, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Activation of the ER stress response protein kinase R (PKR)-like ER kinase and phosphorylation of eukaryotic translation initiator factor 2α led to upregulation of the proapoptotic molecule C/EBP homologous protein and cell death across a panel of DLBCL cell lines independent of cell of origin. Concurrently, targeting UAE led to accumulation of Cdt1, a replication licensing factor, leading to DNA rereplication, checkpoint activation, and cell cycle arrest. MYC oncoprotein sensitized DLBCL cells to UAE inhibition; engineered expression of MYC enhanced while genetic MYC knockdown protected from TAK-243-induced apoptosis. UAE inhibition demonstrated enhanced ER stress and UPR and increased potency compared with bortezomib in DLBCL cell lines. In vivo treatment with TAK-243 restricted the growth of xenografted DLBCL tumors, accompanied by reduced cell proliferation and apoptosis. Finally, primary patient-derived DLBCL cells, including those expressing aberrant MYC, demonstrated susceptibility to UAE inhibition. In sum, targeting UAE may hold promise as a novel therapeutic approach in NHL.

Cyclin-Dependent Kinase-9 Is a Therapeutic Target in MYC-Expressing Diffuse Large B-Cell Lymphoma.

Deregulation of the MYC transcription factor is a key driver in lymphomagenesis. MYC induces global changes in gene expression that contribute to cell growth, proliferation, and oncogenesis by stimulating the activity of RNA polymerases. A key feature in its ability to stimulate RNA Pol II activity is recruitment of pTEFb, an elongation factor whose catalytic core comprises CDK9/cyclin T complexes. Hence, MYC expression and function may be susceptible to CDK9 inhibition. We conducted a pre-clinical assessment of AZ5576, a selective CDK9 inhibitor, in diffuse large B-cell lymphoma (DLBCL). The in vitro and in vivo effects of AZ5576 on apoptosis, cell cycle, Mcl-1, and MYC expression were assessed by flow cytometry, immunoblotting, qPCR and RNA-Seq. We demonstrate that, in addition to depleting Mcl-1, targeting CDK9 disrupts MYC oncogenic function. Treatment with AZ5576 inhibited growth of DLBCL cell lines in vitro and in vivo, independent of cell-of-origin. CDK9 inhibition downregulated Mcl-1 and MYC mRNA transcript and protein in a dose-dependent manner. MYC-expressing cell lines demonstrated enhanced susceptibility to AZ5576. CDK9 inhibition promoted turnover of MYC protein, and decreased MYC phosphorylation at the stabilizing Ser62 residue and downregulated MYC transcriptional targets in DLBCL cells, a finding confirmed in a functional reporter assay, suggesting that CDK9 may govern MYC protein turnover, thus regulating its expression through multiple mechanisms. Our data suggest that targeting CDK9 is poised to disrupt MYC oncogenic activity in DLBCL and provide rationale for clinical development of selective CDK9 inhibitors.