Targeting CDA-Directed Metabolism With 5-Formyl-2'-Deoxycytidine For ALK Inhibitor–Resistant Lung Cancer Therapy

Background: Acquired resistance to inhibitors of anaplastic lymphoma kinase (ALK) is a major clinical challenge for ALK fusion–positive non-small-cell lung cancer (NSCLC). In the absence of secondary ALK mutations, epigenetic reprogramming is one of the main mechanisms of drug resistance as it leads to phenotype switching that occurs during the epithelial-to-mesenchymal transition (EMT). While drug-induced epigenetic reprogramming is believed to alter the sensitivity of cancer cells to anticancer treatments, there is still much to learn about overcoming drug resistance. Methods: We used an in vitro model of ceritinib-resistant NSCLC and employed genome-wide DNA methylation analysis in combination with single-cell (sc) RNA-seq to identify cytidine deaminase (CDA), a pyrimidine salvage pathway enzyme, as a candidate drug target. Molecular biology was used to characterize the role of CDA in drug resistance. Integrated analysis of scRNA-seq and scATAC-seq identified gene regulatory networks in resistant cells. Clinical relevance of CDA was evaluated using TCGA datasets, patient-derived cells, and tumor biopsies. Results: CDA was hypomethylated and upregulated in ceritinib-resistant cells. CDA-overexpressing cells were rarely but definitively detected in the na¨ıve cell population by scRNA-seq, and their abundance increased in the acquired-resistance population. Knockdown of CDA had antiproliferative e↵ects on resistant cells and reversed the EMT phenotype. Treatment with epigenome-related nucleosides such as 5-formyl-2’-deoxycytidine selectively ablated CDA-overexpressing resistant cells via accumulation of DNA damage. Conclusions: Targeting CDA metabolism using epigenome-related nucleosides represents a potential new therapeutic strategy for overcoming ALK-inhibitor resistance in NSCLC.

Epigenetic reprogramming of airway macrophages promotes polarization and inflammation in muco-obstructive lung disease

Lung diseases, such as cystic fibrosis and COPD, are characterized by mucus obstruction and chronic airway inflammation, but their mechanistic link remains poorly understood. Here, we focus on the function of the mucostatic airway microenvironment on epigenetic reprogramming of airway macrophages (AM) and resulting transcriptomic and phenotypical changes. Using a mouse model of muco-obstructive lung disease (Scnn1b-transgenic), we identify epigenetically controlled, differentially regulated pathways and transcription factors involved in inflammatory responses and macrophage polarization. Functionally, AMs from Scnn1b-transgenic mice have reduced efferocytosis and phagocytosis, and excessive inflammatory responses upon lipopolysaccharide challenge, mediated through enhanced Irf1 function and expression. Ex vivo stimulation of wild-type AMs with native mucus impairs efferocytosis and phagocytosis capacities. In addition, mucus induces gene expression changes, comparable with those observed in AMs from Scnn1b-transgenic mice. Our data show that mucostasis induces epigenetic reprogramming of AMs, leading to changes favoring tissue damage and disease progression. Targeting these altered AMs may support therapeutic approaches in patients with muco-obstructive lung diseases.

Duvelisib Promotes Mitochondrial Fusion and Epigenetic Reprogramming to Drive Therapeutic T Cell Persistence and Function

Chronic lymphocytic leukemia (CLL), a cancer of B-lymphocytes, is the most common leukemia in adults. While current frontline therapies for CLL, such as ibrutinib or combination venetoclax and obintuzumab, have significantly improved clinical outcome for patients with treatment naïve CLL and relapsed and refractory CLL (RR-CLL), complete response (CR) rates for RR-CLL patients on ibrutinib remain between 5-14% and 42% for patients on venetoclax and obintuzumab (1, 2). With the advent of chimeric antigen receptor T cells (CART), CR for RR-CLL have increased to around 26-29%, yet this is in sharp contrast to the 70-93% CR achieved in acute lymphocytic leukemia (2). This discrepancy in response is in part due to the inherently immunosuppressive nature of CLL, such that CLL patients are significantly deficient in CD8 co-receptor expressing (CD8+) T cells, including stem cell-like memory T (Tscm) and central memory T (Tcm) cells (2). As the prevalence of Tscm and Tcm cell populations is directly correlated to the in vivo persistence and efficacy of CART, elucidating translatable mechanisms to selectively expand Tscm and Tcm from CLL patients is key to improving the efficacy CART cell therapy for CLL patients. Memory T cell activation, differentiation, and maintenance are processes that are tightly regulated by mitochondrial fusion, fatty acid oxidation, and oxidative phosphorylation (OXPHOS) (3). Moreover, enforcing T cell mitochondrial fusion improves CART cell efficacy against solid tumors (4). As metabolism plays an important role in memory T cell biology, identifying key metabolic pathways that can be targeted ex vivo during CART expansion is of particular interest. To that end, we have shown that dual inhibition of Phosphoinositide 3-Kinase (PI3K) δ/γ isoforms with IPI-145 (duvelisib) during ex vivo T cell manufacturing, preferentially expands CD8+ T cells, including Tscm and Tcm, as well as improves the in vivo persistence (Figure 1A) and cytotoxicity (Figure 1B) of CD19-targeted CART (CD19-CART) (5). To investigate the role of mitochondrial dynamics during ex vivo expansion of duvelisib treated T cell cultures, we stimulated CLL patient-derived T cells with anti-CD3/CD28 beads, re-stimulated T cells on day 9, and harvested T cell cultures on day 15. Immunoblot analysis of day 15 samples indicates that ex vivo duvelisib treatment of CLL patient T cells increases expression of key mitochondrial fusion proteins, mitofusins 1 and 2 (MFN1/2), and decreases serine 637 phosphorylation of mitochondrial fission protein, DRP1, without coincident upregulation of the master regulator of mitochondrial biogenesis, PPARG coactivator 1 alpha (Figure 2A). In addition, duvelisib increased the expression of sirtuins 1 and 3 (SIRT1/3), which have known roles in the post-translational activation of MFN1/2, as well as other epigenetic regulators of memory T cell development and persistence, including FOXO1, TCF1/7, and ID3 (Figure 2B). Taken together, these data suggest that duvelisib promotes mitochondrial fusion and epigenetic reprogramming of T cells during ex vivo expansion. To further interrogate the role of PI3K δ/γ inhibition in mitochondrial dynamics and metabolism, we analyzed T cell cultures following 15 days of duvelisib treatment using a series of extracellular flux and transmission electron microscopy (TEM) experiments. Duvelisib promotes an increase in the total mitochondrial cross-sectional area of both un-transduced and CD19-CAR transduced T cells (Figure 3) and maintains basal, coupled, and spare respiratory capacity of un-transduced T cells on day 15 of expansion (Figure 4). In summary, our data suggest that mitochondrial fusion through MFN1/2 and epigenetic reprogramming facilitate PI3K δ/γ inhibition-mediated ex vivo T cell expansion, where the SIRT1/3-MFN1/2 axis serves as a potential intersection between mitochondrial fusion and epigenetic reprogramming. Figure 1 Figure 1. Disclosures Waller: Verastem Oncology: Consultancy, Research Funding; Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Determinants of Innate Immunity in Visceral Leishmaniasis and Their Implication in Vaccine Development

Leishmaniasis is endemic to the tropical and subtropical regions of the world and is transmitted by the bite of an infected sand fly. The multifaceted interactions between Leishmania, the host innate immune cells, and the adaptive immunity determine the severity of pathogenesis and disease development. Leishmania parasites establish a chronic infection by subversion and attenuation of the microbicidal functions of phagocytic innate immune cells such as neutrophils, macrophages and dendritic cells (DCs). Other innate cells such as inflammatory monocytes, mast cells and NK cells, also contribute to resistance and/or susceptibility to Leishmania infection. In addition to the cytokine/chemokine signals from the innate immune cells, recent studies identified the subtle shifts in the metabolic pathways of the innate cells that activate distinct immune signal cascades. The nexus between metabolic pathways, epigenetic reprogramming and the immune signaling cascades that drive the divergent innate immune responses, remains to be fully understood in Leishmania pathogenesis. Further, development of safe and efficacious vaccines against Leishmaniasis requires a broader understanding of the early interactions between the parasites and innate immune cells. In this review we focus on the current understanding of the specific role of innate immune cells, the metabolomic and epigenetic reprogramming and immune regulation that occurs during visceral leishmaniasis, and the strategies used by the parasite to evade and modulate host immunity. We highlight how such pathways could be exploited in the development of safe and efficacious Leishmania vaccines.

Methylome inheritance and enhancer dememorization reset an epigenetic gate safeguarding embryonic programs

Drastic epigenetic reprogramming is essential to convert terminally-differentiated gametes to totipotent embryos. However, it remains puzzling why post-fertilization global DNA reprogramming occurs only in mammals but not in non-mammalian vertebrates. In zebrafish, global methylome inheritance is however accompanied by sweeping enhancer "dememorization" as they become fully methylated. By depleting maternal dnmt1 using oocyte microinjection in situ, we eliminated DNA methylation in zebrafish early embryos, which died around gastrulation with severe differentiation defects. Strikingly, methylation deficiency leads to extensive derepression of adult tissue-specific genes and CG-rich enhancers, which acquire ectopic TF binding and, unexpectedly, H3K4me3. By contrast, embryonic enhancers are generally CG-poor and evade DNA methylation repression. Hence, global DNA hypermethylation inheritance coupled with enhancer dememorization installs an epigenetic gate that safeguards embryonic programs and ensures temporally ordered gene expression. We propose that "enhancer dememorization" underlies and unifies distinct epigenetic reprogramming modes in early development between mammals and non-mammals.