Reshaping the Tumor Microenvironment of KRASG12D Pancreatic Ductal Adenocarcinoma with combined SOS1 and MEK Inhibition for Improved Immunotherapy Response.

KRAS inhibitors have demonstrated exciting pre-clinical and clinical responses, although resistance occurs rapidly. Here, we investigate the effects of KRAS-targeting therapies on the tumor microenvironment using a library of KRASG12D, p53 mutant, murine PDAC-derived cell lines (KPCY) to leverage immune-oncology combination strategies for long-term tumor efficacy. Our findings show that SOS1 and MEK inhibitors (SOS1i+MEKi) suppressed tumor growth in syngeneic models and increased intra-tumoral CD8+ T cells without durable responses. scRNA-sequencing revealed an increase in inflammatory cancer associated fibroblasts (iCAFs), M2 macrophages, and a decreased dendritic cell quality that ultimately resulted in a highly immunosuppressive microenvironment driven by IL6+ iCAFs. Agonist CD40 treatment was effective to revert macrophage polarization and overcome the lack of mature antigen presenting DCs after SOS1i+MEKi therapy. Treatment increased the overall survival of KPCY tumor-bearing mice. The addition of checkpoint blockade to SOS1i+MEKi combination resulted in tumor free mice with established immune memory. Our data suggests that KRAS inhibition affects myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong anti-tumor effects.

Landscape and Dynamics of Single Immune Cells in Hepatocellular Carcinoma.

The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45+ immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3+ dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3+ DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45+ cell types revealed by this study add new dimensions to the immune landscape of HCC.

The discovery of thienopyridine analogues as potent IkappaB kinase beta inhibitors. Part II.

An SAR study that identified a series of thienopyridine-based potent IkappaB Kinase beta (IKKbeta) inhibitors is described. With focuses on the structural optimization at C4 and C6 of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C4, whereas polar groups with proper orientation at C6 efficiently enhance compound potency. The most potent analogues inhibit IKKbeta with IC50s as low as 40 nM, suppress LPS-induced TNF-alpha production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-kappaB reporter gene assay, demonstrating that they directly interfere with the NF-kappaB signaling pathway.

Discovery and characterization of a substrate selective p38alpha inhibitor.

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.

Optimization of 2-phenylaminoimidazo[4,5-h]isoquinolin-9-ones: orally active inhibitors of lck kinase.

The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.

IKKalpha, IKKbeta, and NEMO/IKKgamma are each required for the NF-kappa B-mediated inflammatory response program.

The IKKbeta and NEMO/IKKgamma subunits of the NF-kappaB-activating signalsome complex are known to be essential for activating NF-kappaB by inflammatory and other stress-like stimuli. However, the IKKalpha subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-kappaB-dependent and -independent functions. In contrast to this generally accepted view of IKKalpha's physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKbeta and NEMO/IKKgamma, IKKalpha is also a global regulator of tumor necrosis factor alpha- and IL-1-responsive IKK signalsome-dependent target genes including many known NF-kappaB targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, beta(2)-microglobulin, IL-1alpha, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-kappaB-dependent target genes were preferentially dependent on IKKalpha or IKKbeta. Constitutive expression of a trans-dominant IkappaBalpha superrepressor (IkappaBalphaSR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-kappaB. A subset of NF-kappaB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-kappaB in established MEFs. Overall, a sizable number of novel NF-kappaB/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-beta and -delta, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (alpha and beta) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-kappaB/IKK-induced gene, suggesting that IKK-mediated NF-kappaB activation can coordinately illicit negative effects on STAT signaling.

Discovery of 2-phenylamino-imidazo[4,5-h]isoquinolin-9-ones: a new class of inhibitors of lck kinase.

An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.