Targeted theranostic photoactivation on atherosclerosis

Background Photoactivation targeting macrophages has emerged as a therapeutic strategy for atherosclerosis, but limited targetable ability of photosensitizers to the lesions hinders its applications. Moreover, the molecular mechanistic insight to its phototherapeutic effects on atheroma is still lacking. Herein, we developed a macrophage targetable near-infrared fluorescence (NIRF) emitting phototheranostic agent by conjugating dextran sulfate (DS) to chlorin e6 (Ce6) and estimated its phototherapeutic feasibility in murine atheroma. Also, the phototherapeutic mechanisms of DS-Ce6 on atherosclerosis were investigated. Results The phototheranostic agent DS-Ce6 efficiently internalized into the activated macrophages and foam cells via scavenger receptor-A (SR-A) mediated endocytosis. Customized serial optical imaging-guided photoactivation of DS-Ce6 by light illumination reduced both atheroma burden and inflammation in murine models. Immuno-fluorescence and -histochemical analyses revealed that the photoactivation of DS-Ce6 produced a prominent increase in macrophage-associated apoptotic bodies 1 week after laser irradiation and induced autophagy with Mer tyrosine-protein kinase expression as early as day 1, indicative of an enhanced efferocytosis in atheroma. Conclusion Imaging-guided DS-Ce6 photoactivation was able to in vivo detect inflammatory activity in atheroma as well as to simultaneously reduce both plaque burden and inflammation by harmonic contribution of apoptosis, autophagy, and lesional efferocytosis. These results suggest that macrophage targetable phototheranostic nanoagents will be a promising theranostic strategy for high-risk atheroma. Graphical abstract

Identification of Targetable Kinases in Idiopathic Pulmonary Fibrosis

Background: Tyrosine kinase activation plays an important role in the progression of pulmonary fibrosis. In this study, we analyzed the expression of 612 kinase-coding and cancer-related genes using next-generation sequencing to identify potential therapeutic targets for idiopathic pulmonary fibrosis (IPF).Methods: Thirteen samples from five patients with IPF (Cases 1-5) and eight samples from four patients without IPF (control) were included in this study. Six of the thirteen samples were obtained from different lung segments of a single patient who underwent bilateral pneumonectomy. Gene expression analysis of IPF lung tissue samples (n=13) and control samples (n=8) was performed using SureSelect RNA Human Kinome Kit. The expression of the selected genes was further confirmed at the protein level by immunohistochemistry (IHC).Results: Gene expression analysis revealed a correlation between the gene expression signatures and the degree of fibrosis, as assessed by Ashcroft score. In addition, the expression analysis indicated a stronger heterogeneity among the IPF lung samples than among the control lung samples. In the integrated analysis of the 21 samples, DCLK1 and STK33 were found to be upregulated in IPF lung samples compared to control lung samples. However, the top most upregulated genes were distinct in individual cases. DCLK1, PDK4, and ERBB4 were upregulated in IPF case 1, whereas STK33, PIM2, and SYK were upregulated in IPF case 2. IHC revealed that these proteins were expressed in the epithelial layer of the fibrotic lesions.Conclusions: We performed a comprehensive kinase expression analysis to explore the potential therapeutic targets for IPF. DCLK1 and STK33 can serve as potential candidate targets for molecular targeted therapy of IPF. In addition, PDK4, ERBB4, PIM2, and SYK may serve as personalized therapeutic targets of IPF.

CRTC1–SS18 Fusion Sarcoma With Aberrant Anaplastic Lymphoma Kinase Expression

Undifferentiated small round cell sarcoma (USRCS) represents a highly heterogeneous group of tumors. A variety of specific gene fusions of USRCS have been reported, including CIC–FOXO4, CIC–NUTM1, BCOR–MAML3, and ZC3H7B–BCOR. Here we report a case of sarcoma harboring a rare recurrent CRTC1–SS18 gene fusion, which was considered as USRCS previously. This sarcoma was composed of nests of small round cells encapsulated in a fibrous stroma. Foci of necrosis and hemorrhage were observed in the tumor. Immunohistochemistry for anaplastic lymphoma kinase showed diffuse positivity. RNA-seq results revealed a chromosomal translocation of CRTC1 gene exon 1 on chromosome 19 with SS18 gene exon 2 on chromosome 18. Thereafter, fluorescence in-situ hybridization confirmed the presence of SS18 gene and CRTC1 gene break-apart, which manifested as the splitting of red and green signals into 2 parts. A previous study showed that CRTC1–SS18 fusion sarcoma and EWSR1–CREB1 fusion angiomatoid fibrous histiocytoma were clustered close in the expression profile. However, whether CRTC1–SS18 fusion sarcomas represent a high malignancy has been a matter of debate. Our study is a worthy addition to the series of rare rearrangements associated with sarcomas and may be of therapeutic relevance.

Identification of pathogenic CDK12 alterations in cell-free DNA (cfDNA) from patients with breast cancer.

1028 Background: Cyclin dependent kinase 12 ( CDK12) has both tumor suppressive and proto-oncogenic potential in metastatic breast cancers (MBC). CDK12 may be an important biomarker and target in MBC. However, a comprehensive genomic analysis of CDK12 alterations from cfDNA in MBC has not been investigated and the genomic impact of CDK12 alterations across the MBC spectrum is unknown. The purpose of this study was to identify the incidence of CDK12 genomic alterations occurring in cfDNA from patients with MBC and elucidate which CDK12 alterations may impact CDK12 kinase activity. Methods: We queried 13,070 MBC samples from the Guardant Health database between April 2019 – November 2020 to identify the incidence of CDK12 alterations detected in cfDNA. We classified each alteration type as: missense mutations, indels, or truncations. Amino acid changes occurring at conserved regions across multiple species were identified. Three-dimensional biochemical in silico analyses with ChimeraX were used to determine which CDK12 alterations may impact CDK12 kinase activity. To gain further biologic insights into CDK12 altered MBC we made associations with CDK12 alterations and co-occurring mutated genes. Results: Nonsynonymous CDK12 alterations from the Guardant Health database were found in 317 samples from a cohort of 13,070 patients indicating an overall incidence of 2.43%. Alterations included: 239 (75.4%) missense mutations; 26 (8.2%) indels; and 52 (16.4%) truncations. We identified 62 alterations within the kinase domain with all occurring at highly conserved regions across species. The most frequent hotspot mutation identified was I76M/T, occurring in 11 unique breast cancers. Three-dimensional analyses indicate that CDK12 alterations within the hinge, HRD, DFG, catalytic spine, and regulatory spine may impact CDK12 kinase activity. The significantly co-occurring mutations from the Guardant Health breast cancer database in samples with CDK12 alterations were ARID1A, APC, RB1, and PTEN. Conclusions: A modest incidence of CDK12 genomic alterations occur in cfDNA from patients with breast cancer. Novel somatic alterations in CDK12 were identified from Guardant Health that were not detected in the public domain. A portion of these occurred at highly conserved regions across species suggesting these specific CDK12 mutations may impact CDK12 kinase expression and be actionable therapeutic targets in breast cancers. Three dimensional analyses of the CDK12 gene further illustrate which specific alterations may induce CDK12 kinase expression or lead to inactivation. Co-occurring mutations reveal a unique genotype associated with CDK12 alterations that may play a biologic role in CDK12-mediated breast cancer pathogenesis. Preclinical studies to determine the prognostic and therapeutic implication of CDK12 alterations in MBC are warranted.

Co-Targeting PIM Kinase and PI3K/mTOR in NSCLC

PIM kinases are constitutively active proto-oncogenic serine/threonine kinases that play a role in cell cycle progression, metabolism, inflammation and drug resistance. PIM kinases interact with and stabilize p53, c-Myc and parallel signaling pathway PI3K/Akt. This study evaluated PIM kinase expression in NSCLC and in response to PI3K/mTOR inhibition. It investigated a novel preclinical PI3K/mTOR/PIM inhibitor (IBL-301) in vitro and in patient-derived NSCLC tumor tissues. Western blot analysis confirmed PIM1, PIM2 and PIM3 are expressed in NSCLC cell lines and PIM1 is a marker of poor prognosis in patients with NSCLC. IBL-301 decreased PIM1, c-Myc, pBAD and p4EBP1 (Thr37/46) and peIF4B (S406) protein levels in-vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in tumor tissue explants. IBL-301 significantly decreased secreted pro-inflammatory cytokine MCP-1. Altered mRNA expression, including activated PIM kinase and c-Myc, was identified in Apitolisib resistant cells (H1975GR) by an IL-6/STAT3 pathway array and validated by Western blot. H1975GR cells were more sensitive to IBL-301 than parent cells. A miRNA array identified a dysregulated miRNA signature of PI3K/mTOR drug resistance consisting of regulators of PIM kinase and c-Myc (miR17-5p, miR19b-3p, miR20a-5p, miR15b-5p, miR203a, miR-206). Our data provides a rationale for co-targeting PIM kinase and PI3K-mTOR to improve therapeutic response in NSCLC.