The translocation of CCTα, the rate-limiting enzyme for phosphatidylcholine synthesis, to nuclear membranes and nuclear lipid droplets results in reversible dephosphorylation of S319 and sustained phosphorylation of Y359+S362. Independent regulation of these phosphosites in the P-domain of CCTα is required for activation on nuclear membranes.

PMD-026 is a first in class, reversible, oral small molecule inhibitor of p90 ribosomal S6 kinase (RSK), a kinase family activated by the MAPK and PDK-1 pathways, which regulate substrates involved in cancer cell proliferation and drug resistance. Specifically, RSK2 has been identified as a major driver in breast cancer (BC). In preclinical studies and a Phase I clinical trial in metastatic BC, PMD-026 demonstrated a good safety profile, making it an attractive candidate for combinations with standard of care therapies. Here in we show that RSK2 is activated in 87% of triple negative breast cancer (TNBC), as well as in other subtypes (80% of ER+/PR+ and 81% of HER2+). In a screen of 28 BC cell lines across a broad mutational spectrum, PMD-026 induced apoptosis in most of the models. Next, to determine the role of PMD-026 in the context of conventional BC treatment, we combined it with standard of care chemotherapies fulvestrant, paclitaxel, or doxorubicin, in vitro and in vivo. PMD-026 synergized with fulvestrant, with combination drug index (CDI) values ranging from 0.73 - 0.92 in the HR+ cell line MCF-7. Likewise, PMD-026 synergized with paclitaxel and doxorubicin in TNBC cell lines with CDI values ranging from 0.40 - 0.84 and 0.58 - 0.92 for paclitaxel and doxorubicin, respectively. Consistent with in vitro screening data, PMD-026 synergized with paclitaxel in vivo in the SUM149PT TNBC xenograft model after 38 days of treatment. The combination inhibited tumor growth by 66% (P < 0.0001), whereas paclitaxel and PMD-026 as single agents inhibited tumor growth by 22% (P = 0.3051) and 41% (P = 0.0041), respectively. The synergy of this combination was further supported by a CDI value of 0.75. Treatment among the groups was well tolerated with no changes in body weight observed. Similarly, the combination of PMD-026 and paclitaxel was synergistic in SUM149PT(48 days, CDI: 0.78) and MDA-MB-231 (28 days, CDI: 0.66) xenografts and additive in a PDx model of metastatic TNBC (18 days, CDI: 0.96), resulting in improved survival. To address the safety of combining PMD-026 and paclitaxel, drug-drug interactions (DDI) were assessed. In cytochrome P450-mediated in vitro metabolism assays, PMD-026 showed weak inhibition of Cyp2C8 and Cyp3A4, the main enzymes responsible for paclitaxel metabolism. To understand whether PMD-026 might alter the metabolism of paclitaxel, this potential DDI was assessed in vivo. Pharmacokinetic analysis of PMD-026 (7 days repeat dosing) combined with paclitaxel (8 mg/kg IV dose Day 1 and Day 7) was evaluated in CD-1 mice, however, PMD-026 did not change the absorption, distribution, or blood levels of paclitaxel. Together, these data support adding PMD-026 to standard of care therapies in breast cancer, as they have demonstrated synergy. In particular, PMD-026 is synergistic with paclitaxel in multiple TNBC models, in which it improves efficacy without added toxicity. Citation Format: Aarthi Jayanthan, My-my Huynh, Jangsoon Lee, Gerrit Los, Lambert Yue, Mary Rose Pambid, Naoto T. Ueno, Sandra E. Dunn. PMD-026, a first in class oral RSK inhibitor, demonstrates synergy when combined with standard of care in breast cancer tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1038.

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Bhargava et al., 2016) that described how we intended to replicate selected experiments from the paper “RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth” (Hatzivassiliou et al., 2010). Here we report the results. We found two unrelated RAF inhibitors, PLX4720 or GDC-0879, selectively inhibited BRAF(V600E) cell proliferation, while the MEK inhibitor, PD0325901, inhibited BRAF(V600E), wild-type RAF/RAS, and mutant RAS cancer cell proliferation, similar to the original study (Figure 1A; Hatzivassiliou et al., 2010). We found knockdown of CRAF, but not BRAF, in mutant RAS cells attenuated the phospho-MEK induction observed after PLX4720 treatment, similar to the original study (Figure 2B; Hatzivassiliou et al., 2010). The original study reported analogous results with GDC-0879, which was not observed in this replication, although unexpected control results confound the interpretation. We also attempted a replication of an assay with recombinant proteins to test the differential effect of RAF inhibitors on BRAF-CRAF heterodimerization (Figure 4A; Hatzivassiliou et al., 2010). Although we were unable to conduct the experiment as planned, we observed differential binding of BRAF by RAF inhibitors; however, it was between BRAF and beads, independent of CRAF. While these data were unable to address whether, under the conditions of the original study, the same observations could be observed, we discuss key differences between the original study and this replication that are important to consider for further experiments. Finally, where possible, we report meta-analyses for each result.

The non-receptor focal adhesion kinase (FAK) is highly expressed in the central nervous system during development, where it regulates neurite outgrowth and axon guidance, but its role in the adult healthy and diseased brain, specifically in Alzheimer’s disease (AD), is largely unknown. Using the 3xTg-AD mouse model, which carries three mutations associated with familial Alzheimer’s disease (APP KM670/671NL Swedish, PSEN1 M146V, MAPT P301L) and develops age-related progressive neuropathology including amyloid plaques and Tau tangles, we describe here, for the first time, the in vivo role of FAK in AD pathology. Our data demonstrate that while site-specific knockdown in the hippocampi of 3xTg-AD mice has no effect on learning and memory, hippocampal overexpression of the protein leads to a significant decrease in learning and memory capabilities, which is accompanied by a significant increase in amyloid β (Aβ) load. Furthermore, neuronal morphology is altered following hippocampal overexpression of FAK in these mice. High-throughput proteomics analysis of total and phosphorylated proteins in the hippocampi of FAK overexpressing mice indicates that FAK controls AD-like phenotypes by inhibiting cytoskeletal remodeling in neurons which results in morphological changes, by increasing Tau hyperphosphorylation, and by blocking astrocyte differentiation. FAK activates cell cycle re-entry and consequent cell death while downregulating insulin signaling, thereby increasing insulin resistance and leading to oxidative stress. Our data provide an overview of the signaling networks by which FAK regulates AD pathology and identify FAK as a novel therapeutic target for treating AD.

Post-homogenization instability of phosphorylation sites in proteins, and the insensitivity and high costs of most proteomics analytical methods have been major barriers in tracking cell signalling networks in minute specimens of cells and tissues. While antibody microarrays have been promising tools for quantifying changes in protein expression and phosphorylation, the interpretation of findings from their applications have been complicated by issues of protein-protein interactions and cross-reactivity with off-target proteins. We used the human A431 cervical carcinoma cell line that over-expresses the epidermal growth factor (EGF) receptor to investigate its signal transduction mechanisms. Chemical cleavage of proteins at cysteine residues at the time of homogenizing the A431 cells permitted preservation of the autophosphorylation of this receptor in response to brief stimulation of these cells with EGF and revealed many downstream signalling events. Most of the EGF-induced changes in protein phosphorylation observed with the antibody microarray were validated by Western blotting with the intended targets, although many of these also false positives that arose from cross-reactivity with the EGF receptor itself. Some false-negatives from blocked epitopes may have stemmed from interactions with SH2 domain-containing adapter proteins that were resistant to chemical cleavage and internal interactions of flanking arginine and lysine residues with phosphosites.