Spen loss drives extra-follicular diffuse large B cell lymphoma with female-specific lethality and TLR pathway therapeutic vulnerabilities

Abstract Diffuse large B cell lymphomas (DLBCL) are the most common lymphoid malignancies in adults. Despite advances in molecular classification, the pathogenesis of DLBCL, particularly of the BN2 subtype, remains poorly understood, which limits the advancement of tailored and more effective therapeutic strategies. BN2-DLBCL are characterized by alterations in BCL6 and NOTCH2, lack an AICDA mutational signature, and are presumed to arise outside germinal centers (GC). Among its defining alterations, truncating mutations in SPEN (SPENTRUNC) are significantly enriched, but the effects and clinical relevance of these alterations remain unexplored. Here, we found that SPENTRUNC likely represent loss-of-function (LOF) events, as they led to reduced SPEN protein levels (p=0.04). Clinically, SPENTRUNC mutations correlated with significantly worse overall survival (OS), especially in non-GCB DLBCL patients (HR: 1.82; p<0.0001). Co-occurring truncating mutations in NOTCH2 (NOTCH2TRUNC), which confer gain-of-function (GOF) effects, further worsened prognosis when in combination with SPENTRUNC (HR: 3.33; p<0.0001). Patients with dual SPENTRUNC/NOTCH2TRUNC (SN2) mutations were also older (p=0.03) and had poorer ECOG performance status (p=0.02), defining a high-risk subgroup urgently needing targeted therapies. To understand how SN2 mutations shape disease biology, we introduced B cell–specific SpenLOF and Notch2GOF mutations in mice. The SN2 genotype led to a cumulative expansion of autoimmune/aged B cells (AiBCs), a hyper-reactive inflammatory B cell subset implicated in autoimmunity and lymphomagenesis. Given the hypothesized extra-follicular origin of BN2-DLBCL, we tested whether AiBCs could arise in SN2 mice lacking Bcl6, which is essential for GC formation. Indeed, AiBC expansion occurred independently of GC formation, as SN2;Bcl6–/– and SN2 mice showed comparable AiBC levels, supporting their extra-follicular derivation. To further evaluate their malignant potential, we assessed clonality and proliferation in SN2 AiBCs versus their wild-type (WT) counterparts. SN2 AiBCs exhibited significantly higher clonality and proliferation (p<0.05). Notably, female SN2 AiBCs showed even greater proliferation (KI67+) than male SN2 AiBCs (p<0.0001), a difference not observed in WT mice. This disproportionate fitness of female SN2 cells translated to a competitive advantage, as shown by bone marrow chimera assays (p=0.04), regardless of the hormonal sex status of the recipient animal. Accordingly, female SN2 mice had significantly reduced survival due to lymphoma development than their male counterparts (OS HR: 13.4; p=0.001), a trend mirrored in human SN2-DLBCL patients (OS HR: 4.14; p=0.07). This sex-bias is of particular interest, as SPEN is known to be essential in X-chromosomal inactivation (XCI), a process happening in females to equilibrate X chromosomal gene dosage to male cells. Although XIST RNA-FISH did not reveal changes in XCI (p=0.2), SN2 lymphomas showed significant hypomethylation of the X chromosome compared to WT B cells and N2 lymphomas (p=5.9e-4). Autosomal regions, in contrast, were hypermethylated (p<2.2e-18), suggesting X-chromosome–specific dysregulation due to SPEN loss. Among X-linked genes, TLR7, a known AiBC driver, emerged as a candidate mediator. Indeed, female SN2 lymphomas expressed higher TLR7 levels than males (p=0.05). To test whether the TLR7 pathway confers an exploitable therapeutic vulnerability, we treated SN2 lymphoma cells with AZ1495, an IRAK1/4 inhibitor acting downstream of TLR7, and found that only female cells showed in vitro and in vivo sensitivity (p<0.05). Efficacy was further confirmed using a female human SN2-DLBCL PDX model (p=0.01), supporting the translational potential of targeting this axis. In summary, SPENTRUNC defines a poor-prognosis marker in DLBCL, and cooperates with NOTCH2TRUNC to drive aggressive, extra-follicular lymphomas via expansion of pathogenic AiBCs. We identify a novel, sex-biased pathogenic mechanism involving X-chromosomal dysregulation and TLR7 overexpression, offering a rationale for precision therapy in BN2-DLBCL.

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