Targeting the tumor microenvironment for treating double refractory chronic lymphocytic leukemia.

The introduction of BTK inhibitors and BCL2 antagonists to the treatment of chronic lymphocytic leukemia has revolutionized therapy and improved patient outcomes. These agents have replaced chemoimmunotherapy as standard of care. Despite this progress, a new group of patients is currently emerging, which has become refractory or intolerant to both classes of agents, creating an unmet medical need. Here, we propose that the targeted modulation of the tumor microenvironment provides new therapeutic options for this group of "double refractory" patients. Furthermore, we outline a sequential strategy for tumor microenvironment-directed combination therapies in CLL that can be tested in clinical protocols.

Role of the tumor microenvironment in CLL pathogenesis.

Chronic lymphocytic leukemia (CLL) cells extensively interact with and depend on their surrounding tumor microenvironment (TME). The TME encompasses a heterogeneous array of cell types, soluble signals, and extracellular vesicles, which contribute significantly to CLL pathogenesis. CLL cells and the TME cooperatively generate a chronic inflammatory milieu, which reciprocally reprograms the TME and activates a signaling network within CLL cells, promoting their survival and proliferation. Additionally, the inflammatory milieu exerts chemotactic effects, attracting CLL cells and other immune cells to the lymphoid tissues. The intricate CLL-TME interactions also facilitate immune evasion and compromise leukemic cell surveillance. We also review recent advances that have shed light on additional aspects that are substantially influenced by the CLL-TME interplay.

Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.

Introduction: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. Methods: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus. Results: Applying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow. Discussion: The two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

LYN kinase programs stromal fibroblasts to facilitate leukemic survival via regulation of c-JUN and THBS1.

Microenvironmental bystander cells are essential for the progression of chronic lymphocytic leukemia (CLL). We have discovered previously that LYN kinase promotes the formation of a microenvironmental niche for CLL. Here we provide mechanistic evidence that LYN regulates the polarization of stromal fibroblasts to support leukemic progression. LYN is overexpressed in fibroblasts of lymph nodes of CLL patients. LYN-deficient stromal cells reduce CLL growth in vivo. LYN-deficient fibroblasts show markedly reduced leukemia feeding capacity in vitro. Multi-omics profiling reveals that LYN regulates the polarization of fibroblasts towards an inflammatory cancer-associated phenotype through modulation of cytokine secretion and extracellular matrix composition. Mechanistically, LYN deletion reduces inflammatory signaling including reduction of c-JUN expression, which in turn augments the expression of Thrombospondin-1, which binds to CD47 thereby impairing CLL viability. Together, our findings suggest that LYN is essential for rewiring fibroblasts towards a leukemia-supportive phenotype.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia.

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles.

Introduction: In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. Methods: We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Results: Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Conclusion: Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.