Lineage-specific proteome remodeling of diverse lung cancer cells by targeted epigenetic inhibitors.

Epigenetic inhibitors exhibit powerful antiproliferative and anticancer activities. However, cellular responses to small-molecule epigenetic inhibition are heterogenous and dependent on factors such as the genetic background, metabolic state, and on-/off-target engagement of individual small-molecule drugs. To determine the mechanisms that drive these heterogeneous cellular responses, we quantified chromatin, proteome, and transcriptome remodeling due to histone deacetylase inhibitor (HDACi) -treated cells derived from diverse genetic backgrounds. We utilized high-throughput sample multiplexed proteomics and integrated intelligent data acquisition methods to map proteomes of cancer cell lines in response to HDACi. We determined cell type-specific and ubiquitous cellular responses based on the quantification of 10,621 total proteins. We then established how coordinated remodeling of the proteome, transcriptome and chromatin state of HDACi treated cancer cells revealed convergent (JUN, MAP2K3, CDKN1A) and divergent (CCND3, ASF1B, BRD7) molecular phenotypes. HDACi-regulated proteins differ greatly across cell lines owing to heterogeneous molecular states of these cell lines. Finally, we demonstrated that HDACi treatment drove a highly cell-type specific response that may in part be explained by cell line-specific off-target drug engagement.

A heterogeneous pharmaco-transcriptomic landscape induced by targeting a single oncogenic kinase.

Over-activation of the epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR-targeted therapies have led to minimal clinical response. While delivery of EGFR inhibitors (EGFRis) to the brain constitutes a major challenge, how additional drug-specific features alter efficacy remains poorly understood. We apply highly multiplex single-cell chemical genomics to define the molecular response of glioblastoma to EGFRis. Using a deep generative framework, we identify shared and drug-specific transcriptional programs that group EGFRis into distinct molecular classes. We identify programs that differ by the chemical properties of EGFRis, including induction of adaptive transcription and modulation of immunogenic gene expression. Finally, we demonstrate that pro-immunogenic expression changes associated with a subset of tyrphostin family EGFRis increase the ability of T-cells to target glioblastoma cells.

ALK Amplification and Rearrangements Are Recurrent Targetable Events in Congenital and Adult Glioblastoma.

PURPOSE: Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. EXPERIMENTAL DESIGN: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. RESULTS: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0-80 years). Recurrent as well as novel ALK fusions (LRRFIP1-ALK, DCTN1-ALK, PRKD3-ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. CONCLUSIONS: These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs. See related commentary by Mack and Bertrand, p. 2567.

4-Hydroxybutyrate Promotes Endogenous Antimicrobial Peptide Expression in Macrophages.

IMPACT STATEMENT: This study evaluated the biological activity of hydroxylated derivatives of butyrate as inductors of antimicrobial peptides (AMPs) in murine bone marrow-derived macrophages in vitro. A differential modulation of AMP expression by the hydroxylated derivatives of butyrate is shown. The ability of sodium 4-hydroxybutyrate to upregulate AMP expression through a histone deacetylase inhibitory-independent mechanism, and to promote increased resistance to bacterial contamination in vivo are also shown. The findings provide an alternative for prevention of bacterial contamination of implanted biomaterials. Functionalization of biomaterials with hydroxylated derivatives of butyrate can enhance the endogenous antimicrobial activity of the immune system through increased production of AMPs by host cells, thus providing protection against bacterial contamination.

The Effect of Mechanical Loading Upon Extracellular Matrix Bioscaffold-Mediated Skeletal Muscle Remodeling.

Mounting evidence suggests that site-appropriate loading of implanted extracellular matrix (ECM) bioscaffolds and the surrounding microenvironment is an important tissue remodeling determinant, although the role at the cellular level in ECM-mediated skeletal muscle remodeling remains unknown. This study evaluates crosstalk between progenitor cells and macrophages during mechanical loading in ECM-mediated skeletal muscle repair. Myoblasts were exposed to solubilized ECM bioscaffolds and were mechanically loaded at 10% strain, 1 Hz for 5 h. Conditioned media was collected and applied to bone marrow-derived macrophages followed by immunolabeling for proinflammatory M1-like markers and proremodeling M2-like markers. Macrophages were subjected to the same loading protocol and their secreted products were collected for myoblast migration, proliferation, and differentiation analysis. A mouse hind limb unloading volumetric muscle loss model was used to evaluate the effect of loading upon the skeletal muscle microenvironment after ECM implantation. Animals were sacrificed at 14 or 180 days. Isometric torque production was tested and tissue sections were immunolabeled for macrophage phenotype and muscle fiber content. Results show that loading augments the ability of myoblasts to promote an M2-like macrophage phenotype following exposure to ECM bioscaffolds. Mechanically loaded macrophages promote myoblast chemotaxis and differentiation. Lack of weight bearing impaired muscle remodeling as indicated by Masson's Trichrome stain. Isometric torque was significantly increased following ECM implantation when compared to controls, a response not present in the hind limb-unloaded group. This work provides an important mechanistic insight of the effects of rehabilitation upon ECM-mediated remodeling and could have broader implications in clinical practice, advocating multidisciplinary approaches to regenerative medicine, emphasizing rehabilitation.