Cell-intrinsic Effects Research Articles

Abstract B011: MTA-cooperative PRMT5 inhibition (BMS-986504) induces MTAP del tumor cell-intrinsic immune evasion and regulation

Abstract Homozygous deletion of MTAP gene (MTAP del) occurs in ∼10% of all cancers. BMS- 986504 is a second-generation MTA-cooperative PRMT5 inhibitor designed to preferentially bind to the PRMT5-MTA complex which accumulates in MTAP del cancer cells and has demonstrated clinical proof-of-concept (POC) for selectively inhibiting PRMT5 activity in MTAP del patients as monotherapy. While immunotherapy (IO) has increased patient survival in many cancers and is now the standard of care in first and later lines, most patients ultimately relapse. Thus, there remains an unmet need to improve response for these patients receiving IO. BMS-986504 in combination with anti- PD1 has the potential to increase efficacy, but there is a limited understanding of the interaction between MTAP/PRMT5 biology and PD-1 blockade. Here, we evaluated the BMS-986504-induced immunological and cell killing effects on MTAP del tumor cells and T cells to understand potential mechanisms to support BMS-986504 combination opportunities with anti-PD1 therapy. Using a panel of human PBMC-derived T cells, we first demonstrate that non-activated T cells and T cells activated via anti-CD3/CD28 are insensitive to BMS-986504-induced cell killing at in vitro concentrations equivalent to clinical exposure, supporting the significantly improved selectivity margin of BMS- 986504 relative to first-generation PRMT5 inhibitors that showed T cell toxicity in vitro and on-target dose-limiting hematological toxicity in the clinic. Since previous studies have suggested that MTAP wild-type cells may be affected by MTA secreted from surrounding MTAP del tumors, we assessed T cell viability in the presence of MTA. We found that inactivated T cells maintain insensitivity to BMS-986504 treatment with or without extracellular MTA, but T cells activated via anti-CD3/CD28 are partially sensitive to BMS-986504-induced killing in the presence of MTA only. Using flow cytometry, we characterized the surface expression of immune-related markers on tumor cells treated with BMS-986504 and identified that BMS-986504 dose-dependently increases PD-L1 expression on MTAP del tumor cells, suggesting a potential immune evasion mechanism. Next, using human PBMC-derived T cell and tumor cell co-culture assays, we evaluated the ability of BMS-986504 and anti-PD1 combination to mediate both tumor-intrinsic killing and anti-tumor immune activation in MTAP del cancer cell lines. Taken together, we provide translational proof-of-concept data demonstrating that BMS- 986504 induces tumor cell-intrinsic immunological effects in MTAP del tumors, while having limited impact on T cell viability. This data supports the rationale for BMS- 986504 and IO combination, which may potentially lead to anti-tumor immune activation and improved outcome for MTAP del patients. Citation Format: Josephine Hai, Stephanie Xavier, Sangeeth George, Lars Engstrom, Pete Olson, Tyler Simpson, Ming Lei, Benjamin Chen. MTA-cooperative PRMT5 inhibition (BMS-986504) induces MTAP del tumor cell-intrinsic immune evasion and regulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B011.

Stimulating Soluble Guanylyl Cyclase with the Clinical Agonist Riociguat Restrains the Development and Progression of Castration-Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is incurable and fatal, making prostate cancer the second-leading cancer-related cause of death for American men. CRPC results from therapeutic resistance to standard-of-care androgen deprivation (AD) treatments, through incompletely understood molecular mechanisms, and lacks durable therapeutic options. Here, we identified enhanced soluble guanylyl cyclase (sGC) signaling as a mechanism that restrains CRPC initiation and growth. Patients with aggressive, fatal CRPC exhibited significantly lower serum levels of the sGC catalytic product cyclic GMP (cGMP) compared to their castration-sensitive stage. In emergent castration-resistant cells isolated from castration-sensitive prostate cancer (CSPC) populations, the obligate sGC heterodimer was repressed via methylation of its beta subunit. Genetically abrogating sGC complex formation in CSPC cells promoted evasion of AD-induced senescence and concomitant castration-resistant tumor growth. In established castration-resistant cells, the sGC complex was present but in a reversibly oxidized and inactive state. Subjecting CRPC cells to AD regenerated the functional complex, and co-treatment with riociguat, an FDA-approved sGC agonist, evoked redox stress-induced apoptosis. Riociguat decreased castration-resistant tumor growth and increased apoptotic markers, with elevated cGMP levels correlating significantly with lower tumor burden. Riociguat treatment reorganized tumor vasculature and eliminated hypoxic tumor niches, decreasing CD44+ tumor progenitor cells and increasing the radiosensitivity of castration-resistant tumors. Thus, this study showed that enhancing sGC activity can inhibit CRPC emergence and progression through tumor cell-intrinsic and extrinsic effects. Riociguat can be repurposed to overcome CRPC, with noninvasive monitoring of cGMP levels as a marker for on-target efficacy.

Acid ceramidase expression reduces IFNγ secretion by mouse CD4+ T cells and is crucial for maintaining B-cell numbers in mice.

Acid ceramidase (Ac) is a lysosomal enzyme catalyzing the generation of sphingosine from ceramide, and Ac inhibitors are currently being investigated as potential cancer therapeutics. Yet, the role of the Ac in immune responses, particularly anti-viral immunity, is not fully understood. To investigate the impact of Ac expression on various leukocyte populations, we generated a tamoxifen-inducible global knockout mouse model for the Ac (iAc-KO). Following tamoxifen administration to healthy mice, we extracted primary and secondary lymphoid organs from iAc-KO and wild-type (wt) littermates and subsequently performed extensive flow cytometric marker analysis. In addition, we isolated CD4+ T cells from the spleen and lymph nodes for sphingolipid profiling and restimulated them in vitro with Dynabeads™ Mouse T-activator CD3/CD28. Intracellular cytokine expression (FACS staining) was analyzed and secreted cytokines detected in supernatants. To study cell-intrinsic effects, we established an in vitro model for iAc-KO in isolated CD4+ T and B cells. For CD4+ T cells of iAc-KO versus wt mice, we observed reduced Ac activity, an increased ceramide level, and enhanced secretion of IFNγ upon CD3/CD28 costimulation. Moreover, there was a marked reduction in B cell and plasma cell and blast numbers in iAc-KO compared to wt mice. To study cell-intrinsic effects and in line with the 3R principles, we established in vitro cell culture systems for iAc-KO in isolated B and CD4+ T cells. Our findings pinpoint to a key role of the Ac in mature B and antibody-secreting cells and in IFNγ secretion by CD4+ T cells.

IL-27 promotes pathogenic T cells in a mouse model of Sjögren's disease

Sjögren's disease (SjD) is a chronic autoimmune disease characterized by focal lymphocytic inflammation in lacrimal and salivary glands. We recently identified IL-27 as a requisite signal for the spontaneous SjD-like manifestations in nonobese diabetic (NOD) mice. Here, we define T cell-intrinsic effects of IL-27 in lacrimal gland disease in NOD mice. IL-27 receptor was required by both CD4 T effector (Te) cells and CD8 T cells to mediate focal inflammation. Intrinsic IL-27 signaling was associated with PD-1 and ICOS expressing T follicular helper (Tfh)-like CD4 Te cells within lacrimal glands, including subsets defined by CD73 or CD39 expression. CD8 T cells capable of IL-27 signaling also expressed PD-1 with subsets expressing ICOS and CD73 demonstrating a T follicular cytotoxic (Tfc)-like cell phenotype and others expressing a CD39hi exhausted-like phenotype. These findings suggest IL-27 is a key early signal driving a follicular-type response in lacrimal gland inflammation in NOD mice.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

IntroductionPatients with pediatric low-grade gliomas (pLGGs), the most common primary brain tumors in children, can often benefit from MAPK inhibitor (MAPKi) treatment. However, rapid tumor regrowth, also referred to as rebound growth, may occur once treatment is stopped, constituting a significant clinical challenge.MethodsFour patient-derived pediatric glioma models were investigated to model rebound growth in vitro based on viable cell counts in response to MAPKi treatment and withdrawal. A multi-omics dataset (RNA sequencing and LC-MS/MS based phospho-/proteomics) was generated to investigate possible rebound-driving mechanisms. Following in vitro validation, putative rebound-driving mechanisms were validated in vivo using the BT-40 orthotopic xenograft model.ResultsOf the tested models, only a BRAFV600E-driven model (BT-40, with additional CDKN2A/Bdel) showed rebound growth upon MAPKi withdrawal. Using this model, we identified a rapid reactivation of the MAPK pathway upon MAPKi withdrawal in vitro, also confirmed in vivo. Furthermore, transient overactivation of key MAPK molecules at transcriptional (e.g. FOS) and phosphorylation (e.g. pMEK) levels, was observed in vitro. Additionally, we detected increased expression and secretion of cytokines (CCL2, CX3CL1, CXCL10 and CCL7) upon MAPKi treatment, maintained during early withdrawal. While increased cytokine expression did not have tumor cell intrinsic effects, presence of these cytokines in conditioned media led to increased attraction of microglia cells in vitro.ConclusionTaken together, these data indicate rapid MAPK reactivation upon MAPKi withdrawal as a tumor cell intrinsic rebound-driving mechanism. Furthermore, increased secretion of microglia-recruiting cytokines may play a role in treatment response and rebound growth upon withdrawal, warranting further evaluation.

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract 4307: Quantifying the cell-intrinsic and cell-extrinsic growth effects of cancer-causing mutations in vivo

Abstract Adaptive driver phenotypes in cancer must confer either a cell-intrinsic (birth minus death) or cell-extrinsic (field) effect on growth. These effects can be summarized by an r/K logistic growth curve, whereby cell-intrinsic effects manifest as a change in the early, exponential growth constant (r) of a tumor population while cell-extrinsic effects alter the long-term ‘carrying capacity’ (K). Previously, we developed a novel genetically-engineered mouse model that tracks tumor growth dynamics with unprecedented precision via Tumor Barcoding, termed TuBa-seq1. Here, we quantify the growth trajectories of thousands of Kras-driven lung tumors over eight months. Median tumor size is best-described by a Gomp-Ex growth law (i.e. Nν/K where ν → +0), which also characterizes advanced-stage human tumors2. Carrying capacities of individual isogenic tumors varied by over four orders of magnitude, suggesting that microenvironmental heterogeneity profoundly impacts growth dynamics. Our approach further allowed us to track the growth effects of eleven canonical tumor suppressor gene knockouts introduced via multiplexed CRISPR/Cas genome-engineering. All but one of these secondary mutations augmented either the exponential growth rate or the carrying capacity of the tumor. However, only three tumor suppressor losses increased both growth parameters (Rb1, Setd2, and Stk11). Hence, the growth benefits of most cancer-causing mutations are missed when growth is measured only at a single time point. Clinically, we find that r-promoting and K-promoting mutations interact synergistically to create the most aggressive tumors in human patients (HR = 1.71, P-value &amp;lt; 10-10). Collectively, these results support a universal r/K framework for tumor growth and progression.

Abstract 5367: Single cell resolution spatial transcriptomics reveals genetic driver and TME interaction in clear cell renal cell carcinoma

Abstract BAP1 mutations are associated with increased T cell and tumor-associated macrophage infiltration in clear cell renal cell carcinoma (ccRCC) and are predictive of poor prognosis. BAP1 mutations have also been found to be associated with T-cell infiltration in peritoneal mesothelioma and uveal melanoma. Because BAP1 mutations are known to be associated with aggressive disease and poor survival, we hypothesize that these mutations lead to a more immunosuppressive microenvironment. Using single nucleus RNA sequencing and high-resolution spatial transcriptomics, we confirmed that BAP1 mutant tumors are immune hot with a high fraction of Treg, exhausted CD8+ T cells, CD4+ T cells, and macrophages. At the same time, these tumors have a lower proportion of endothelial cells and pericytes. Evident of pro-tumor microenvironment we have found BAP1 mutations to be associated with the upregulation of SPP1, CD163, and MARCO immunosuppressive genes in tumor-associated macrophages. Here we propose a mechanism by which BAP1-mutant cancer cells induce an immunosuppressive microenvironment: through activation of the complement system and TGF-β signaling to attract macrophages. Taken together, our results suggest that BAP1 mutations contribute to poor ccRCC outcomes not only through cell-intrinsic effects, but also by promoting infiltration of Tregs and tumor-associated macrophages and polarizing infiltrated macrophages towards immunosuppressive phenotype. Citation Format: Ilya Strunilin, Wagma Caravan, Atieh Abedin-Do, Andrew Houston, Yize Li, Yizhe Song, Song Cao, Chia-Kuei Mo, Siqi Chen, Russel Pachynski, Feng Chen, Li Ding. Single cell resolution spatial transcriptomics reveals genetic driver and TME interaction in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5367.

Abstract 4259: A novel human model of lung squamous cell carcinoma to deconvolve the cell-intrinsic effects of key dysregulated pathways

Abstract Tractable, patient relevant models are needed to investigate the essential components of normal cell transformation. We developed a unique in vitro model of early lung squamous cell carcinoma (LUSC) genetic alterations and driver pathway dysregulation using genetically modified primary human bronchial basal cells (hBCs) from three independent donors. The co-operation of ubiquitous and early LUSC alterations (TP53 and CDKN2A loss) and commonly deregulated pathways including squamous differentiation (SOX2), PI3K signaling (PTEN) and the oxidative stress response (KEAP1) was investigated using a combinatorial approach, culminating in eight unique hBC mutants harboring cumulative pathway alterations in a TP53-/-/CDKN2A-/- background. Phenotypes indicative of mutant hBC transformation were investigated using proliferation assays, organotypic cultures and anchorage independent growth assays. Organotypic cultures revealed LUSC pathway alterations to drive dramatic changes in airway histology, ranging from a normal epithelium to severe dysplasia. Furthermore, these analyses confirmed the role of SOX2-overexpression in initiating transformation by inducing squamous cell commitment and anchorage independent growth. We also identified a co-operation of SOX2-overexpression with the oxidative stress response and PI3K pathways to drive more advanced lesions in-vivo and in-vitro. Importantly, we were able to map changes in the expression of immunomodulatory factors with the pathway dysregulation. We demonstrated that this strategy constitute an alternative and patient-relevant system to model LUSC and identify genotype-phenotype correlations of clinical relevance. Citation Format: Julia Ogden, Robert Sellers, Anthony Oojageer, Sudhakar Sahoo, Anshuman Chaturvedi, Carlos Lopez-Garcia. A novel human model of lung squamous cell carcinoma to deconvolve the cell-intrinsic effects of key dysregulated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4259.

Mitf regulates gene expression networks implicated in B cell homeostasis, germinal center responses, and tolerance.

The microphthalmia transcription factor Mitf has been shown to regulate B cell activation and tolerance. However, the underlying B cell-specific mechanisms responsible, and those that distinguish Mitf from closely related Mitf/TFE (MiT) transcription factors Tfe3, Tfeb, and Tfec, remain obscure. Two complementary mouse models of Mitf and MiT deficiency were used: the Mitfmi-vga9/mi-vga9 systemic loss-of-function mutation, and B-cell specific MiT family inactivation via transgenic expression of a trans-dominant negative (TDN) protein (TDN-B). These models were employed to identify MiT family candidate target genes and pathways. Both models displayed spontaneous splenomegaly coincident with elevated plasma cell numbers, autoantibody titers, and proteinuria. These abnormalities appeared dependent on T helper cells, but independent of other non-B cell intrinsic effects of systemic Mitf inactivation. MiT inactivation in B cells augmented aspects of lupus-like autoimmune disease on the C57BL/6-Faslpr/lpr background. In both models, RNAseq of ex vivo resting B cells showed transcriptional upregulation of genes that control cell cycle, germinal center responses, and plasma cell differentiation. Among the genes strongly upregulated in both models were Socs6, Isp53 (Baiap1), S1pR2, and IgG2b/c. Mitf null B cells, but not TDN-B cells, showed evidence of type I interferon dysregulation. These studies clarify Mitf's role as 1) a key regulator of a B cell intrinsic germinal center program that influences self-tolerance through novel target genes, and 2) a regulator of systemic inflammatory processes that can impact the B cell microenvironment. This distinction of Mitf's function from that of related MiT transcription factors advances our understanding of B cell regulation and autoimmunity.

Immune checkpoints between epithelial-mesenchymal transition and autophagy: A conflicting triangle

Inhibitory immune checkpoint (ICP) molecules are pivotal in inhibiting innate and acquired antitumor immune responses, a mechanism frequently exploited by cancer cells to evade host immunity. These evasion strategies contribute to the complexity of cancer progression and therapeutic resistance. For this reason, ICP molecules have become targets for antitumor drugs, particularly monoclonal antibodies, collectively referred to as immune checkpoint inhibitors (ICI), that counteract such cancer-associated immune suppression and restore antitumor immune responses. Over the last decade, however, it has become clear that tumor cell-associated ICPs can also induce tumor cell-intrinsic effects, in particular epithelial-mesenchymal transition (EMT) and macroautophagy (hereafter autophagy). Both of these processes have profound implications for cancer metastasis and drug responsiveness. This article reviews the positive or negative cross-talk that tumor cell-associated ICPs undergo with autophagy and EMT. We discuss that tumor cell-associated ICPs are upregulated in response to the same stimuli that induce EMT. Moreover, ICPs themselves, when overexpressed, become an EMT-inducing stimulus. As regards the cross-talk with autophagy, ICPs have been shown to either stimulate or inhibit autophagy, while autophagy itself can either up- or downregulate the expression of ICPs. This dynamic equilibrium also extends to the autophagy-apoptosis axis, further emphasizing the complexities of cellular responses. Eventually, we delve into the intricate balance between autophagy and apoptosis, elucidating its role in the broader interplay of cellular dynamics influenced by ICPs. In the final part of this article, we speculate about the driving forces underlying the contradictory outcomes of the reciprocal, inhibitory, or stimulatory effects between ICPs, EMT, and autophagy. A conclusive identification of these driving forces may allow to achieve improved antitumor effects when using combinations of ICIs and compounds acting on EMT and/or autophagy. Prospectively, this may translate into increased and/or broadened therapeutic efficacy compared to what is currently achieved with ICI-based clinical protocols.

Abstract A002: Genotype-directed synthetic cytotoxicity of ATR inhibition with radiotherapy

Abstract The DNA damage response is crucial for the efficacy of radiotherapy (RT), prompting efforts to target this pathway with radiosensitizers to improve outcomes. Early clinical trials evaluating this approach, however, have failed to yield evidence of benefit in unselected populations. Here we demonstrate that a genotype-directed combination of DNA repair inhibition and RT can unleash the significant therapeutic benefit of this approach in pre-clinical models and present first experience from a Phase I/II trial (NCT05566574) testing this concept. Using CRISPR/Cas9, we created isogenic murine models (Atm-/- and WT) in 4T1 breast and CT26 colorectal cancer models, and then studied the synergy between camonsertib and RT across genotypes. In vitro, camonsertib radio-sensitized both Atm-/- and WT models, but was more potent and synergistic in Atm-/- models. In the orthotopically implanted 4T1 model, the combination of camonsertib (30 mg/kg, 3 days on; 4 days off) with RT (20Gy x 1) significantly delayed tumor growth in Atm-/- tumors compared to WT (p &amp;lt; 0.001; n=10 mice per group). Overall survival (OS) also improved in mice with Atm-/- tumors (20% alive at 150 days) vs. WT (0% alive; p &amp;lt; 0.001; log-rank test). Similar results were observed using other RT dose-fractionations (4Gy x 5 daily, 6Gy x 3, 8Gy x 2) with camonsertib. In the flank-implanted CT26 model, Atm-/- tumors had markedly improved response to combination therapy with 7/8 mice achieving a complete response (CR) compared to only 1/8 WT tumors (p&amp;lt;0.001). OS was also significantly improved with 87% of mice with CT26 Atm-/- tumors cured compared to 0% of CT26 WT tumors (p &amp;lt; 0.001). To evaluate the genotype-specificity for synergy from combination therapy, we performed similar experiments in a 4T1 Brca1-/- model, a key mediator of homologous recombination, and observed an absence of synergy, consistent with our hypothesis. Analysis of the microenvironment of 4T1 Atm-/- treated with combination therapy by flow cytometry did not reveal significant increases in CD4 or CD8 T-cells compared to vehicle nor increases in activated CD8 T-cells (Ki67+, PD1+, or GzmB+) suggesting the therapeutic benefit was primarily mediated by cell intrinsic effects. Similarly, re-challenge experiments in cured mice did not identify strong evidence of immunologic memory, again suggesting a modest immunologic role for efficacy of combination therapy in this setting. Based on these results, we initiated a Phase I/II study of camonsertib with RT (4Gy x 5) in patients with metastatic cancer that require palliative RT and have mutations in ATM (pathogenic or variant of unknown significance). The first two patients with pathogenic ATM mutant tumors enrolled in the Phase I portion of the study have demonstrated metabolic CR to combination therapy on PET scan. Overall, our findings suggest that genotype-directed concurrent radio-sensitization could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments. Further research to test this hypothesis is ongoing. Citation Format: Victor Ng, Sonali Sinha, Ardijana Novaj, Jennifer Ma, Niamh McDermott, Ana Longhini, Rui Gardner, Ezra Rosen, Simon Powell, Diana Mandelker, Atif Khan, Jeremy Setton, Anne Roulston, Stephen Morris, Maria Koehler, Nancy Lee, Jorge Reis-Filho, Nadeem Riaz. Genotype-directed synthetic cytotoxicity of ATR inhibition with radiotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A002.

Pathogenic Mechanisms of DDX41 Mutations in the Development of Myeloid Malignancies

DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele (most commonly p.A500fs or p.D140fs, depending on the ethnicity) is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms have not fully been elucidated. To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles. Vav1-Cre mediated homozygous deletion of Ddx41 resulted in embryonic lethality, suggesting that Ddx41 is indispensable for normal hematopoiesis. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 ( Ddx41 +/+), heterozygous Ddx41 KO( Ddx41 +/-), homozygous Ddx41 KO( Ddx41 -/-), heterozygous for the Ddx41 R525H mutation ( Ddx41 R525H/+), or hemizygous for the Ddx41 R525H mutation( Ddx41 R525H/-), in which expression of the mutant allele was induced by tamoxifen administration. First, we assessed cell intrinsic effects of these Ddx41 alleles, using noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were transplanted with BM from Ddx41 -/- or Ddx41 R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF), which was not observed in mice transplanted with BM from Ddx41 +/+, Ddx41 +/- or Ddx41 R525H/+ mice. By contrast, the mice transplanted with Ddx41 +/- or Ddx41 R525H/+ BM showed significantly reduced WBC counts and anemia in long-term observation in both primary and serial transplantations. Some of the Ddx41 +/- or Ddx41 R525H/+ BM-transplanted mice exhibited MDS-like phenotypes, showing ineffective hematopoiesis with evidence of erythroid dysplasia. Transcriptome analysis revealed that stem cells (Kit +Sca-1 -Lin low cells) derived from Ddx41 R525H/- BM-transplanted mice exhibited a significant upregulation of genes involved in innate immunity, including interferon stimulated genes, compared with stem cells derived from Ddx41 +/+ BM-transplanted mice. In addition, snoRNA and ribosomal genes were significantly deregulated in stem cells from Ddx41 -/- and Ddx41 R525H/- BM-transplanted mice, which could result in abnormal ribosome biogenesis and protein synthesis in Ddx41 mutant cells. We also assessed the reconstitution capacity of whole BM cells from different Ddx41 mutant mice in competitive transplantation experiments. The donor chimerism of Ddx41 -/- and Ddx41 R525H/- BM-transplanted mice in PB was markedly reduced compared to Ddx41 +/+ BM-transplanted mice. In contrast, Ddx41 +/- and Ddx41 R525H/+ BM-transplanted micedid not show significant changes in competitive bone marrow reconstitution compared to Ddx41 +/+ BM-transplanted mice. Given that the MDS clones bearing DDX41 R525H somatic alleles are typically observed as a small subclone in patients, we next co-transplanted Ddx41 +/+- and Ddx41 525H/--derived BM cells with Ddx41 +/+- or Ddx41 +/--derived BM cells at the ratio of 1:9. Recipient mice showed significantly reduced WBC counts when Ddx41 +/+- or Ddx41 +/- were co-transplanted with Ddx41 525H/- -derived BM, suggesting that Ddx41 525H/--derived hematopoietic cells have negative effect on normal hematopoiesis. In summary, compound biallelic loss-of function and R525 alleles led to severe BM failure. Monoallelic Ddx41 loss-of function and R525H knock-in alleles, by contrast, are compatible with hematopoiesis but associated with an impairment of hematopoiesis and the development of MDS with ageing, where activated innate immunity, impaired RNA metabolism and ribosome functions may play important roles.

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

STING-driven activation of T cells: relevance for the adoptive cell therapy of cancer

Adoptive cell therapy (ACT) can successfully treat hema-topoietic cancers but lacks efficacy against solid tumors. This is due to insufficient T cell infiltration, high tumor heterogeneity, frequent antigen loss with subsequent tumor escape, and the immunosup-pressive tumor microenvironment (TME). Alternative methods to boost the anticancer efficacy of adoptively transferred cells are ac-tively pursued. Among adjuvants that are utilized to stimulate anti-cancer immune responses, ligands of the stimulator of interferon genes (STING) pathway have received increasing attention. STING activation can trigger dendritic cell (DC) activation and endogenous immune responses, thereby preventing tumor escape. Activation of the STING pathway in the context of ACT was accordingly associated with improved T cell trafficking and persistence in the TME com-bined with the reduced presence of immunosuppressive cells. Re-cent findings also suggest cell-intrinsic effects of STING ligands on T cells. Activation of the STING signaling pathway was in this regard shown to enhance effector functions of CD4+ and CD8+ T cells, sug-gesting that the STING signaling could be exploited to harness T cell anticancer functions. In this review, we will discuss how the STING signaling can be used to enhance the anticancer efficacy of ACT.

Identification of cell-intrinsic effects of mutated circulating cells due to clonal hematopoiesis in patients with heart failure

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and is associated with worse prognosis in patients with heart failure with reduced ejection fraction (HFrEF). Circulating immune cells derived from patients with HFrEF harboring CHIP mutations show enhanced proinflammatory signatures. However, it is unclear, whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 patients with HFrEF. Using an improved protocol to detect mutant cells on a single cell level (MutDetect-Seq), as illustrated in figure 1A, we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells). DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. Specifically, DNMT3A mutant monocytes exhibit altered gene expression profiles associated with inflammation and phagolysosome function (figure 1B). In addition, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards effector cells and away from a naïve T cell state. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes. Conclusion This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HFrEF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte as well as the skewing of mutated T cells towards effector cells provide novel insights into how CHIP might promote worse prognosis in heart failure patients.MutDetect-seq

Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.

Tumor Cell-Intrinsic PD-L1 Effects on Radiation-Induced Locoregional Antitumor Immunity

Targeting PD-L1 is a beneficial strategy to reinvigorate antitumor immunity, however variable response and resistance are challenging and suggest the need for multimodality approaches. Tumor cell-intrinsic PD-L1 signals also regulate non-canonical pathogenic pathways that may impact treatment resistance. Ionizing radiation (IR) can induce antitumor immunity and has demonstrated therapeutic synergy with immunotherapy in some cases, however tumor-driven immunologic mechanisms affecting clinical outcomes remain incompletely understood. In this study, we investigated the impact of tumor cell-intrinsic PD-L1 signals on IR-induced locoregional immune response and tumor control. We used orthotopic B16-F10 melanoma (WT-B16) and 4T1 triple negative breast cancer (WT-4T1) murine tumor models, as well as PD-L1 disabled variants (KO) generated by CRISPR/Cas9, implanted bilaterally. IR (10 Gy) was targeted at one tumor alone to evaluate both direct and indirect IR effects based on tumor PD-L1 status. We evaluated response by tumor volume (TV) measurements, flow cytometry of tumor-infiltrating lymphocytes (TILs) and tumor draining lymph nodes (TDLNs) in both irradiated and unirradiated compartments, and granzyme B (GZB) PET imaging to assess functional in vivo changes. Chemokine-based multiplex assays were used to assess cell lines receiving IR (4Gy) and ex vivo tumor lysates and serum. IR-induced local tumor control was not significantly affected based on tumor PD-L1 status, however deactivation of tumor cell PD-L1 enhanced IR-induced regional tumor control. Unirradiated WT tumors in mice harboring irradiated KO but not irradiated WT tumors demonstrated a significant mean reduction in TV with instances of complete distant tumor regression. PET imaging demonstrated a nearly 2-fold higher concentration of GZB in KO versus WT tumors, in line with known locally immunosuppressive effects of tumor PD-L1. Remarkably, GZB levels were >1.5-fold higher in unirradiated WT tumors in mice harboring an irradiated KO versus WT tumor, which correlated with a 50% increase in PD-1+CD8+ T cells. Higher levels of CD62+CD44- naïve CD4+ (4-fold) and CD8+ (2-fold) memory T cells were seen in TDLNs of irradiated KO versus WT tumors. Cytokine levels positively correlated with immune recruitment and activation status, as CXCL10, CCL2 and CCL5 were significantly upregulated in PD-L1 KO versus WT tumors cells. Results from this study demonstrate cell-intrinsic PD-L1 inhibits IR-induced locoregional immune activation and frequency of regional tumor control, with clinical implications including therapeutic targeting of tumor cell-intrinsic PD-L1 signals to enhance IR-induced immunogenicity, utility of IR based on tumor PD-L1 status particularly in the metastatic setting, and immunotherapy combinations. Future studies investigating mechanisms of resistance to IR-induced immune activation to enhance responsiveness are warranted.

Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes

Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro.