The role of Galpha13 signaling in suppression of lymphoma

Division Of Basic Sciences - Nci

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Abstract

Aim 1- Mechanism of Galpha13-mediated tumor suppression in mucosal germinal center B cells. B cells in GCs are among the most highly proliferative somatic cells in the body and are strictly confined to the GC niche that forms at the center of the B cell follicle. In previous work, we defined a tumor suppressive pathway involving the G-protein, Galpha13 (GNA13) that is frequently inactivated in GC-derived DLBCL (Muppidi et al. Nature, 2014). We showed that Galpha13 signaling confined GC B cells to their niche and prevented access of normally non-recirculatory GC B cells to lymph and blood. Additionally, we found that loss of Galpha13 in B cells promoted an accumulation of GC B cell numbers in mesenteric lymph nodes (mLNs) and that aged animals lacking Galpha13 developed GC-derived DLBCL in mLNs. However, it was not clear what microenvironmental cues within the mLN were required to promote outgrowths of Galpha13-deficient GC B cells and subsequent tumorigenesis. Nor was it clear by what molecular mechanism Galpha13 signaling suppresses cell accumulation at this site. We found that Galpha13-deficiency supported GC B cell accumulation in the mLN via increased proliferation that was driven by mTORC1 and MYC. mTORC1 and MYC expression in GC B cells are thought to be primarily driven by T cell help. However, we found the advantage for Galpha13-deficient GC B cells was not dependent on T cells. Additionally, although microbiota can modulate GC selective processes in the mLN, outgrowths of Galpha13-deficient GC B cells occurred in the absence of gut microbiota. Instead, Galpha13-deficient GC B cells were selectively dependent on dietary nutrients likely due to greater access to factors arriving via gut lymphatics. We found that diet-derived glutamine differentially supported proliferation and MYC expression in Galpha13-deficient GC B cells in the mLN. These findings suggest that GC confinement limits the effects of dietary glutamine on GC dynamics in mucosal tissues and that Galpha13 pathway mutations can exploit these processes to promote gut tropism of aggressive lymphoma. This work established that dietary cues can be important drivers of GC-derived DLBCL in mucosal tissues (Nguyen et al. Nature Immunology, 2024). Future work will be aimed at understanding the molecular mechanism of Galpha13-mediated suppression of mTORC1 and MYC protein using both in vitro and in vivo approaches. Aim 2- Defining the cell-of-origin for genetic classes of DLBCL. Early efforts to deconvolute DLBCL heterogeneity used gene expression profiling to identify disease subsets. These efforts identified subsets with expression similarity to GC B cells (GCB-DLBCL) or activated B cells (ABC-DLBCL), however, even within these subsets there remained significant heterogeneity both in terms of clinical outcomes and genetic alterations. Additionally, approximately 15% of DLBCL cases remained "unclassified" by gene expression profiling. Newer efforts to categorize DLBCL based on the landscape of genetic alterations found in an individual tumor have established novel genetic subclasses. The MCD genetic class of DLBCL is highly related to ABC-DLBCL. MCD is enriched for gain-of-function mutations in MYD88 and CD79B but also harbors recurrent mutations in a large number of genes whose function has not been characterized. MCD is thought to be derived from cells that have exited, or are in the process of exiting the GC, whose terminal differentiation into plasma cells has been blocked. More recent work has suggested that differentiation into memory B cells may be an important step in the pathogenesis of MCD, but this has not been formally demonstrated in vivo. The BN2 genetic class is enriched in cases of "unclassified" DLBCL and is defined by gain-of-function mutations in NOTCH2 and translocations involving BCL6. The molecular basis of the genetic co-association between BCL6 and NOTCH2 alterations is unclear, and a cell-of-origin in this subclass has not yet been described. 2.1 The role of spontaneous splenic GCs in MCD. MCD DLBCL carries a poor prognosis following conventional immunochemotherapy, harbors a distinct set of genetic alterations and has unique molecular vulnerabilities. However, despite this extensive molecular characterization, the pre-malignant cell-of-origin for MCD DLBCL remains unclear. To better define the development of MCD in vivo, we analyzed mice carrying up to 4 hallmark genetic lesions found in MCD DLBCL. B cell immune responses and GCs are typically studied in the context of immunization or infection. However, GCs can form in the spleens of unimmunized mice. These spontaneous splenic GCs have unique dependencies compared to GCs at other anatomical sites. We discovered that expression of MCD-associated genetic changes strongly promoted an accumulation of B cells in spontaneous splenic GCs. These pre-malignant GC B cells showed multiple novel dependencies similar to those observed in MCD. Young mice carrying all four genetic lesions showed a greater than 50-fold expansion of spontaneous splenic GCs and ultimately developed DLBCL in the spleen with age (Pindzola et al. Blood, 2022). Thus, we identified expansion spontaneous splenic GC B cells as key step in the pathogenesis of MCD DLBCL. Other groups have proposed that memory B cell differentiation is critical for MCD lymphomagenesis. Our current work aims to clearly defining the role of memory B cells in tumor formation in mice harboring MCD-associated genetic changes. 2.2 The molecular basis of the co-association of Notch2 gain-of-function mutations and BCL6 translocations. The biology of the BN2 genetic subclass of DLBCL is largely unknown. We have developed novel genetically engineered mouse models that: 1) conditionally express a DLBCL-associated mutation from the endogenous Notch2 locus and 2) conditionally over-express BCL6. We are assessing how expression of each allele alters B cell differentiation in vivo when expressed in all B cells or only after B cells have entered the GC reaction at steady state and following immunization. We are assessing whether co-expression of mutant Notch2 and BCL6 is sufficient to drive lymphomagenesis in vivo. We have developed a strategy to rapidly and conditionally delete single or multiple genes in GCB and GC-derived cells in vivo with CRISPR/Cas9. We are determining which genetic events found in BN2 DLBCL in humans, can cooperate with mutant Notch2 and BCL6 to promote pre-malignant B cell expansions and lymphomagenesis in vivo.