Apoptotic Lymphoma Cells Evoke a Pro-Senescent Stromal Signal That Limits Lymphoma Development.

Cancer entities frequently exhibiting constitutive Myc expression, such as aggressive B-cell lymphomas, typically display significant amounts of apoptotic cell death. So far, cellular senescence as another cell-autonomous oncogene-inducible safeguard program has been recognized in RAS/BRAF-driven scenarios but not as a bona fide Myc-evoked anti-cancer mechanism. Understanding how oncogenic Myc may provoke not only apoptosis but cellular senescence as a failsafe mechanism to counter tumor development has broad implications for the clinical presentation and therapeutic strategies in frequently Myc-driven lymphoma entities such as Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL).

Using the Burkitt's like Eμ-myc transgenic mouse lymphoma model, we show here that cellular senescence serves as another crucial anti-neoplastic barrier during Myc-driven tumorigenesis in addition to apoptosis. Eμ-myc lymphomas harbor a substantial fraction of senescent tumor cells, that stain positive for histone H3 lysine 9 (H3K9)-trimethylation. Lymphomas lacking the H3K9 methyltransferase Suv39h1 display no senescence and develop significantly faster, although apoptosis is not affected by Suv39h1 deficiency. While Myc, unlike other Ras-type oncogenes, shows rather modest pro-senescent activity in vitro, we identified the cytostatic cytokine TGF-β as the main paracrine senescence trigger in vivo. When neutralizing TGF-β action during Myc-driven lymphomagenesis utilizing a secretable TGF-β receptor II ecto-domain, senescence is completely blunted and tumor latency is significantly shortened. We identify macrophages, but not lymphoma cells, as the main source of exogenous TGF-β, that is secreted upon phagocytosis of apoptotic lymphoma cells. Lymphomas harboring a Bcl2-mediated apoptotic block presented with a much lower frequency of both infiltrating macrophages and senescent cells in vivo, suggesting that there is a functional link between cell-autonomous Myc-triggered apoptosis and non-cell-autonomous, macrophage-induced senescence. Both pharmacological suppression of TGF-β production in macrophages via the angiotensin-converting enzyme (ACE) inhibitor lisinopril and depletion of macrophages in Eμ-myc lymphoma-harboring mice by systemic exposure to clodronate resulted in a profound reduction of senescence, thereby underscoring the important role for tumor-infiltrating macrophages in TGF-β-mediated senescence in vivo. We recapitulated components of such a mechanism in human aggressive B-cell lymphomas, a frequently Myc-activated entity where TGF-β1 signaling has previously been identified as a component of the prognostically favourable “stromal-1” signature (Lenz-G et al., NEJM, 2008). A panel of 30 DLBCL samples was sub-divided based on Ki67 immunoreactivity into a very high proliferation (Ki67^hi; ≥80% Ki67-positive cells) and a lower proliferation (Ki67^lo; <80% Ki67-positive cells) group. Ki67^lo samples exhibited a higher frequency of H3K9me3-positive cells, indicative of cellular senescence. Importantly, the Ki67^lo group also presented with a higher fraction of apoptotic cells, more lymphoma-infiltrating macrophages, and a stronger reactivity for the TGF-β signaling mediator Smad3-P, thereby representing a subgroup in DLBCL that displays features highly reminiscent of the macrophage-derived mechanism of senescence induction.

Our study expands the relevance of oncogene-induced senescence to Myc-driven cancers and demonstrates that different tumor suppressor programs - such as apoptosis and senescence - are enforced in an interdependent fashion between the tumor- and non-malignant stroma cells during lymphomagenesis. Utilizing the Eμ-myc transgenic mouse lymphoma model and furthermore supported by evidence from human aggressive B-cell lymphoma samples, this study establishes a novel network of heterotypic cell-cell interactions within a tumor in which apoptotic tumor cells induce a paracrine response in non-malignant bystander cells that limits lymphomagenesis by cellular senescence. Given the anti-cancer relevance of senescence and the demonstrated inducibility of senescence by a non-DNA damaging cytokine, such as TGF-β, these findings open the exciting perspective to utilize Suv39h1/H3K9me3-mimicking approaches for future cancer therapies.

No relevant conflicts of interest to declare.