PFS24 as a prognostic milestone in patients with newly diagnosed primary CNS lymphoma

High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation has significantly improved overall survival (OS) in primary central nervous system lymphoma (PCNSL). However, early identification of long-term survivors remains a challenge. Progression-free survival at 24 months (PFS24) has emerged as a key prognostic marker in diffuse large B-cell lymphoma, but its relevance in PCNSL is still unclear. In this retrospective multicenter study, we analyzed data from 146 newly diagnosed, transplant-eligible PCNSL patients treated with MATRix-like regimens across 14 hospitals. With a median follow-up of 48 months, the 2-year PFS and OS rates were 50.4% and 65.6%, respectively. Of the 139 patients evaluable for PFS24-analysis, 51.1% reached PFS24, with a subsequent 5-year OS of 96.7%. Of note, the annual hazard rate for progression and death decreased to under 5% after 24 months, remaining stable thereafter. The patients who failed to reach PFS24 had a median OS of only 6.0 months. Key predictors of PFS failure included impaired Karnofsky performance status and treatment dose-reduction. In conclusion, PFS24 was identified as an important prognostic marker in PCNSL. Patients who achieve PFS24 have a favorable prognosis, whereas those who do not face poor outcomes and require innovative treatment approaches. This insight could aid in risk stratification and support the use of PFS24 as a surrogate endpoint in clinical trials

Research for All: Building a Diverse Researcher Community for the All of Us Research Program

OBJECTIVES: The NIH All of Us Research Program (All of Us) is engaging a diverse community of more than 10 000 registered researchers using a robust engagement ecosystem model. We describe strategies used to build an ecosystem that attracts and supports a diverse and inclusive researcher community to use the All of Us dataset and provide metrics on All of Us researcher usage growth. MATERIALS AND METHODS: Researcher audiences and diversity categories were defined to guide a strategy. A researcher engagement strategy was codeveloped with program partners to support a researcher engagement ecosystem. An adapted ecological model guided the ecosystem to address multiple levels of influence to support All of Us data use. Statistics from the All of Us Researcher Workbench demographic survey describe trends in researchers\u27 and institutional use of the Workbench and publication numbers. RESULTS: From 2022 to 2024, some 13 partner organizations and their subawardees conducted outreach, built capacity, or supported researchers and institutions in using the data. Trends indicate that Workbench registrations and use have increased over time, including among researchers underrepresented in the biomedical workforce. Data Use and Registration Agreements from minority-serving institutions also increased. DISCUSSION: All of Us built a diverse, inclusive, and growing research community via intentional engagement with researchers and via partnerships to address systemic data access issues. Future programs will provide additional support to researchers and institutions to ameliorate All of Us data use challenges. CONCLUSION: The approach described helps address structural inequities in the biomedical research field to advance health equity

Improved Recovery from Liver Fibrosis by Crenolanib

Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-β, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas β-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats

Improved Recovery from Liver Fibrosis by Crenolanib

Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-β, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas β-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats.</jats:p

Lysosomal Membrane Permeabilization Sensitizes Ctss-Hyperactive Tumors to BCL2-Targeting Therapies

Hyperactivity of the cysteine protease cathepsin S (CTSS) -either through Y132 mutations or amplification/overexpression- is a recurrent alteration in follicular lymphoma (FL) and promotes tumor growth by inducing a supportive immune microenvironment (Bararia et al, 2020). Of note, patients with CTSS-hyperactive FL had better outcomes with standard therapies, suggesting that CTSS-hyperactivity can sensitize tumors to treatment. CTSS hyperactivity has also been reported in other B cell lymphomas (BCLs) (Dheilly et al, 2020) and solid cancers (Olson & Joyce, 2015). CTSS is mainly localized intralysosomally but can be released into the cytosol by lysosomal membrane permeabilization (LMP). Low level LMP can occur spontaneously (e.g., during cell division) and can be enhanced by treatment. Unlike other cathepsins, cytosolically released CTSS maintains its enzymatic activity at non-acidic pH. Thus, we aimed to (i) identify the determinants of the cytosolic CTSS activity, (ii) determine its impact on the regulation of apoptosis, and (iii) study LMP as a therapeutic approach for CTSS-hyperactive tumors. First, we accrued biochemical, functional, and clinical data supporting the role of cystatin B (CSTB) as a clinically relevant endogenous CTSS inhibitor in BCLs. Through unbiased and complementary proteomics (BioID2 labelling and co-IP followed by LC-MS/MS) we identified CSTB as a direct CTSS-interacting protein (8-fold enriched in the BCL cell line Karpas422 engineered to express CTSS wild type (WT) or Y132D vs CTSS knock-out (KO), P=0.0002). Single-cell RNA-Seq of primary FL (N=10) showed significantly higher CSTB expression in FL cells compared to normal B cells ( P=0.004). Moreover, shRNA mediated knock-down (k/d) of CSTB increased the overall cathepsin activity in BCL cell lines (N=8) by 1.5 to 5.5-fold, most notably in CTSS-hyperactive cells ( Fig A, top). We next employed LMP-inducing tool compounds (LLOMe) and clinically used drugs or analogs (desipramine, hexamethylene amiloride) to release cathepsins into the cytosol. CTSS-hyperactive Karpas422 were significantly more sensitive to LMP-inducing treatments compared to native cells (1.5 to 10-fold reduction of IC50). Importantly, CTSS hyperactivity and CSTB k/d increased LMP-mediated cell killing ( Fig A, bottom). Thus, the cytosolic CTSS/CSTB interaction determines the net cytosolic cathepsin activity and sensitivity of cells to undergo LMP-induced cell death. Next, we hypothesized that LMP-induced cytosolic CTSS hyperactivity could prime BCLs towards apoptosis. We used BH3 profiling to functionally quantify the dependencies and interactions of BCL2 family members in BCLs with and without CTSS hyperactivity. In Karpas422 cells expressing CTSS Y132D, LMP increased overall apoptotic priming and dependencies on the anti-apoptotic proteins MCL-1 (delta priming &gt;30 % at 10 µM, P=0.04), BCL-xL (&gt;45 % at 10 µM, P=0.0006) and BCL2 (&gt; 45 % at 0.5 and 1 µM, P=0.0001). We hypothesized that BCL2 family members are proteolytically cleaved by cytosolic CTSS. Indeed, e.g., BCL2 protein levels were 2.5 to 3.5-fold lower in LLOMe-treated Karpas422 cells with CTSS-hyperactivity compared to CTSS KO, and CSTB k/d further decreased BCL2 levels. To validate CTSS-mediated cleavage of BCL2, we purified FLAG-tagged BCL2 and CTSS WT and Y132D. CTSS WT efficiently cleaved BCL2 in vitro &lt;1 hour at the top ranked predicted cleavage site and the reaction rate increased 1.3-fold for CTSS Y132D. Finally, we hypothesized that LMP sensitizes cells to BCL2-targeting therapies ( Fig B). The combination of LLOMe-induced LMP and the BCL2 inhibitor venetoclax (VEN) showed increased cytotoxicity in CTSS-hyperactive Karpas422 cells compared to monotherapy and CSTB k/d enhanced this phenotype ( Fig A, bottom). We assessed cathepsin activities and generated dose-response curves for VEN with and without LLOMe-induced LMP in 15 primary CLL samples. Thereof, 12 samples had intermediate or high cathepsin activities and LLOMe-induced LMP increased their sensitivity to VEN, including a VEN-resistant sample in which the IC50 decreased to &lt;3 nM. In summary, we show that CSTB is a functionally relevant inhibitor that determines the net activity of LMP-released cytosolic CTSS. Furthermore, LMP-inducing therapies may be a promising approach to sensitize CTSS-hyperactive tumors towards apoptosis by proteolytic cleavage of BCL2 family members

CHOP but Not Bendamustine Reverses <i>EZH2</i> Y641 Mutation Induced MHC-I/II Loss in Human Lymphoma Models

Abstract Chemotherapy combined with anti-CD20 antibodies is the standard treatment for patients with symptomatic advanced stage follicular lymphoma (FL). We have previously shown that EZH2 gene mutations were predictive of differential efficacy of the chemotherapy backbone within the GALLIUM trial (NCT01332968; Jurinovic, ASH 2019). Specifically, patients with EZH2 mutant FL had significantly longer progression free survival with CHOP-based immunochemotherapies as compared to patients with EZH2 wild type FL. In contrast, the EZH2 mutation status was not predictive of treatment outcome in patients receiving bendamustine-based regimens. The underlying biology is unclear. Mutations in EZH2, a histone methyltransferase, occur in 25-30% of FL and mostly affect the Y641 residue within the catalytic domain, resulting in more efficient conversion of H3K27me2 to H3K27me3. First, we tested differences in chemosensitivity in lymphoma cell lines that harbor the FL-hallmark translocation t(14;18)[BCL2/IGH] and intrinsic EZH2 mutations (Karpas422, OCI-Ly1, SU-DHL4, DB) or no EZH2 mutation (SU-DHL16, WSU-FSCCL, OCI-Ly8, OCI-Ly19). Treatment with CHOP (4-hydroperoxycyclophosphamide (4-HC), doxorubicin, vincristine, and prednisone), alone or in combination, or bendamustine revealed marked differences in IC50 for cell viability (CellTiter Glo) and apoptosis (Annexin V) between cell lines, but no correlation with EZH2 mutation status. To better control for cell line specific effects, we stably expressed EZH2 Y641N or wild type (WT) in the EZH2 WT cell line SU-DHL16. EZH2 mutant cells indeed showed significantly increased H3K27me3 levels compared to EZH2 WT cells. However, we did not observe differences in global cellular phenotypes, including cell proliferation and cell cycle phases. Furthermore, IC50 for cell viability and apoptosis with CHOP and bendamustine treatment were not significantly different. As we could not identify differences in direct cytotoxic responses, we hypothesized that EZH2 mutations might indirectly affect treatment efficacy, by altering the interaction of FL cells with their tumor microenvironment (TME) in response to chemotherapy. In a mouse model, Ennishi et al. had previously shown that Ezh2 mutant lymphomas have reduced MHC expression and T-cell infiltrates (Cancer Discovery, 2019). Here, we could show that expression of mutant EZH2 in human SU-DHL16 cells also leads to almost complete MHC-I loss by flow cytometry (Fig A). MHC-I loss was fully reversible when cells were treated with increasing doses of tazemetostat, a specific EZH2 inhibitor, or interferon-gamma. Interestingly, treatment with 4-HC, prednisone, doxorubicin and CHOP also resulted in increasing restoration of MHC-I expression in EZH2 mutant cells, while bendamustine (and vincristine alone) had no impact on MHC expression (Fig B,C). Next, we used a fully human B-cell co-culture model (modified from Caeser et al., Nat Comm 2019) for validation and functional studies. Mirroring the TME-dependence of FL, germinal-center (GC) B-cells from human tonsils immortalized by transduction with BCL2 and MYC absolutely require follicular dendritic cell (FDC) support plus IL21 and CD40L for sustained growth. Stable expression of EZH2 Y641N in these GC-B-cells led to increased H3K27me3 levels as compared to EZH2 WT and empty vector (ev) controls, but did not result in FDC+IL21/CD40L independent growth. Again, we observed significantly lower MHC-I/II expression on EZH2 mutant cells (Fig D). Importantly, we show that EZH2 mutation-induced MHC loss resulted in reduced CytoStim-stimulated conjugate formation when cells were co-cultured with autologous CD4 T-cells isolated from the same tonsils (Fig E). Finally, treatment with doxorubicin but not bendamustine resulted in significant upregulation of MHC-I/II on EZH2 mutant GC-B cells (Fig F). Co-culture experiments with autologous CD4 and CD8 T-cells with and without doxorubicin, CHOP and bendamustine treatment are ongoing to analyze the EZH2 mutation-specific chemotherapy effects on T-cell activation, recruitment, and T-cell mediated killing. In conclusion, our data indicates that the particular chemosensitivity of EZH2 mutant FL to CHOP is not the result of differences in direct cytotoxicity, but -unlike bendamustine- is rather mediated by restoring EZH2 mutation-induced MHC-I/II loss, thereby potentially promoting cytotoxic T-cell responses. Figure 1 Figure 1. Disclosures Hodson: Astra Zeneca: Research Funding. von Bergwelt: Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe &amp; Dohme: Honoraria, Research Funding, Speakers Bureau. Subklewe: MorphoSys: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Janssen: Consultancy; Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau. Weigert: Janssen: Speakers Bureau; Roche: Research Funding; Epizyme: Membership on an entity's Board of Directors or advisory committees. </jats:sec

PFS24 as a prognostic milestone in patients with newly diagnosed primary CNS lymphoma

High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation has significantly improved overall survival (OS) in primary central nervous system lymphoma (PCNSL). However, early identification of long-term survivors remains a challenge. Progression-free survival at 24 months (PFS24) has emerged as a key prognostic marker in diffuse large B-cell lymphoma, but its relevance in PCNSL is still unclear. In this retrospective multicenter study, we analyzed data from 146 newly diagnosed, transplant-eligible PCNSL patients treated with MATRix-like regimens across 14 hospitals. With a median follow-up of 48 months, the 2-year PFS and OS rates were 50.4% and 65.6%, respectively. Of the 139 patients evaluable for PFS24-analysis, 51.1% reached PFS24, with a subsequent 5-year OS of 96.7%. Of note, the annual hazard rate for progression and death decreased to under 5% after 24 months, remaining stable thereafter. The patients who failed to reach PFS24 had a median OS of only 6.0 months. Key predictors of PFS failure included impaired Karnofsky performance status and treatment dose-reduction. In conclusion, PFS24 was identified as an important prognostic marker in PCNSL. Patients who achieve PFS24 have a favorable prognosis, whereas those who do not face poor outcomes and require innovative treatment approaches. This insight could aid in risk stratification and support the use of PFS24 as a surrogate endpoint in clinical trials. © The Author(s) 2025

PARP14 is a novel target in STAT6 mutant follicular lymphoma

AbstractThe variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of tumor cells and complex interactions within the tumor microenvironment (TME). IL-4 producing follicular helper T cells (TFH) are critical components of the FL TME. Binding of IL-4 to IL-4R on FL cells activates JAK/STAT signaling. We identified STAT6 mutations (STAT6MUT) in 13% of FL (N = 33/258), all clustered within the DNA binding domain. Gene expression data and immunohistochemistry showed upregulation of IL-4/STAT6 target genes in STAT6MUT FL, including CCL17, CCL22, and FCER2 (CD23). Functionally, STAT6MUT was gain-of-function by serial replating phenotype in pre-B CFU assays. Expression of STAT6MUT enhanced IL-4 induced FCER2/CD23, CCL17 and CCL22 expression and was associated with nuclear accumulation of pSTAT6. RNA sequencing identified PARP14 -a transcriptional switch and co-activator of STAT6- among the top differentially upregulated genes in IL-4 stimulated STAT6MUT lymphoma cells and in STAT6MUT primary FL cells. Quantitative chromatin immunoprecipitation (qChIP) demonstrated binding of STAT6MUT but not STAT6WT to the PARP14 promotor. Reporter assays showed increased IL-4 induced transactivation activity of STAT6MUT at the PARP14 promotor, suggesting a self-reinforcing regulatory circuit. Knock-down of PARP14 or PARP-inhibition abrogated the STAT6MUT gain-of-function phenotype. Thus, our results identify PARP14 as a novel therapeutic target in STAT6MUT FL.</jats:p