The TLK-ASF1 histone chaperone pathway plays a critical role in IL-1ß-mediated AML progression.

ABSTRACT: Identifying and targeting microenvironment-driven pathways that are active across acute myeloid leukemia (AML) genetic subtypes should allow the development of more broadly effective therapies. The proinflammatory cytokine interleukin-1ß (IL-1ß) is abundant in the AML microenvironment and promotes leukemic growth. Through RNA-sequencing analysis, we identify that IL-1ß-upregulated ASF1B (antisilencing function-1B), a histone chaperone, in AML progenitors compared with healthy progenitors. ASF1B, along with its paralogous protein ASF1A, recruits H3-H4 histones onto the replication fork during S-phase, a process regulated by Tousled-like kinase 1 and 2 (TLKs). Although ASF1s and TLKs are known to be overexpressed in multiple solid tumors and associated with poor prognosis, their functional roles in hematopoiesis and inflammation-driven leukemia remain unexplored. In this study, we identify that ASF1s and TLKs are overexpressed in multiple genetic subtypes of AML. We demonstrate that depletion of ASF1s significantly reduces leukemic cell growth in both in vitro and in vivo models using human cells. Using a murine model, we show that overexpression of ASF1B accelerates leukemia progression. Moreover, Asf1b or Tlk2 deletion delayed leukemia progression, whereas these proteins are dispensable for normal hematopoiesis. Through proteomics and phosphoproteomics analyses, we uncover that the TLK-ASF1 pathway promotes leukemogenesis by affecting the cell cycle and DNA damage pathways. Collectively, our findings identify the TLK1-ASF1 pathway as a novel mediator of inflammatory signaling and a promising therapeutic target for AML treatment across diverse genetic subtypes. Selective inhibition of this pathway offers potential opportunities to intervene effectively, address intratumoral heterogeneity, and ultimately improve clinical outcomes in AML.

Leukemic mutation FLT3-ITD is retained in dendritic cells and disrupts their homeostasis leading to expanded Th17 frequency.

Dendritic cells (DC) are mediators between innate and adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for acute myeloid leukemia (AML) patients. Having a shared myeloid lineage it can be difficult to distinguish bone fide DCs from AML tumor cells. To date, there is little information on the effects of FLT3-ITD in DC biology. To further elucidate this relationship we utilized CITE-seq technology in combination with flow cytometry and multiplex immunoassays to measure changes to DCs in human and mouse tissues. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with AML to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet- phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells in the bone marrow and spleen compartments. Ex vivo stimulation of CD4+ T cells also showed increased Th17 phenotype in AML mice. Moreover, co-culture of AML mouse-derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes and their downstream effects on Th polarization.

Boosting immunity: synergistic antiviral effects of luteolin, vitamin C, magnesium and zinc against SARS-CoV-2 3CLpro.

SARS-CoV-2 was first discovered in 2019 and has disseminated throughout the globe to pandemic levels, imposing significant health and economic burdens. Although vaccines against SARS-CoV-2 have been developed, their long-term efficacy and specificity have not been determined, and antiviral drugs remain necessary. Flavonoids, which are commonly found in plants, fruits, and vegetables and are part of the human diet, have attracted considerable attention as potential therapeutic agents due to their antiviral and antimicrobial activities and effects on other biological activities, such as inflammation. The present study uses a combination of biochemical, cellular, molecular dynamics, and molecular docking experiments to provide compelling evidence that the flavonoid luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one) has antiviral activity against SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) that is synergistically enhanced by magnesium, zinc, and vitamin C. The IC50 of luteolin against 2 µM 3CLpro is 78 µM and decreases 10-fold to 7.6 µM in the presence of zinc, magnesium, and vitamin C. Thermodynamic stability analyses revealed that luteolin has minimal effects on the structure of 3CLpro, whereas metal ions and vitamin C significantly alter the thermodynamic stability of the protease. Interactome analysis uncovered potential host-virus interactions and functional clusters associated with luteolin activity, supporting the relevance of this flavone for combating SARS-CoV-2 infection. This comprehensive investigation sheds light on luteolin's therapeutic potential and provides insights into its mechanisms of action against SARS-CoV-2. The novel formulation of luteolin, magnesium, zinc, and vitamin C may be an effective avenue for treating COVID-19 patients.

Mapping the proteogenomic landscape enables prediction of drug response in acute myeloid leukemia.

Acute myeloid leukemia is a poor-prognosis cancer commonly stratified by genetic aberrations, but these mutations are often heterogeneous and fail to consistently predict therapeutic response. Here, we combine transcriptomic, proteomic, and phosphoproteomic datasets with ex vivo drug sensitivity data to help understand the underlying pathophysiology of AML beyond mutations. We measure the proteome and phosphoproteome of 210 patients and combine them with genomic and transcriptomic measurements to identify four proteogenomic subtypes that complement existing genetic subtypes. We build a predictor to classify samples into subtypes and map them to a "landscape" that identifies specific drug response patterns. We then build a drug response prediction model to identify drugs that target distinct subtypes and validate our findings on cell lines representing various stages of quizartinib resistance. Our results show how multiomics data together with drug sensitivity data can inform therapy stratification and drug combinations in AML.

Targeting CCL2/CCR2 Signaling Overcomes MEK Inhibitor Resistance in Acute Myeloid Leukemia.

PURPOSE: Emerging evidence underscores the critical role of extrinsic factors within the microenvironment in protecting leukemia cells from therapeutic interventions, driving disease progression, and promoting drug resistance in acute myeloid leukemia (AML). This finding emphasizes the need for the identification of targeted therapies that inhibit intrinsic and extrinsic signaling to overcome drug resistance in AML. EXPERIMENTAL DESIGN: We performed a comprehensive analysis utilizing a cohort of ∼300 AML patient samples. This analysis encompassed the evaluation of secreted cytokines/growth factors, gene expression, and ex vivo drug sensitivity to small molecules. Our investigation pinpointed a notable association between elevated levels of CCL2 and diminished sensitivity to the MEK inhibitors (MEKi). We validated this association through loss-of-function and pharmacologic inhibition studies. Further, we deployed global phosphoproteomics and CRISPR/Cas9 screening to identify the mechanism of CCR2-mediated MEKi resistance in AML. RESULTS: Our multifaceted analysis unveiled that CCL2 activates multiple prosurvival pathways, including MAPK and cell-cycle regulation in MEKi-resistant cells. Employing combination strategies to simultaneously target these pathways heightened growth inhibition in AML cells. Both genetic and pharmacologic inhibition of CCR2 sensitized AML cells to trametinib, suppressing proliferation while enhancing apoptosis. These findings underscore a new role for CCL2 in MEKi resistance, offering combination therapies as an avenue to circumvent this resistance. CONCLUSIONS: Our study demonstrates a compelling rationale for translating CCL2/CCR2 axis inhibitors in combination with MEK pathway-targeting therapies, as a potent strategy for combating drug resistance in AML. This approach has the potential to enhance the efficacy of treatments to improve AML patient outcomes.