Briefly, the cells were washed twice in 1 PBS and trypsinized following established protocols. HCC827 cells was 0.1 M and 15 M, respectively. While Rociletinib or Ocimertinib inhibited the parental H1975 cells with GI50 doses Lenvatinib mesylate of 0.18 M, the Ocimertinib-resistant pools of H1975 cells had a GI50 dose of 12 M. The GI50 dose for Rociletinib-resistant H1975 sublines ranged from 4.5-8.0 M. CFM-4 and its novel analog CFM-4.16 attenuated growth of the parental and TKI-resistant NSCLC cells. CFMs activated p38/JNKs, inhibited oncogenic cMet and Akt kinases, while CARP-1 depletion blocked NSCLC cell growth inhibition by CFM-4.16 or Erlotinib. CFM-4.16 was synergistic with B-Raf-targeting in NSCLC, triple-negative breast cancer, and renal cancer cells. A nano-lipid formulation (NLF) of CFM-4.16 in combination with Sorafenib elicited a superior growth inhibition of xenografted tumors derived from Rociletinib-resistant H1975 NSCLC cells in part by stimulating CARP-1 and apoptosis. These findings support therapeutic potential Lenvatinib mesylate of CFM-4.16 together with B-Raf targeting in treatment of TKI-resistant NSCLCs. CARP-1 homolog lst 3 functioned as an antagonist of EGFR signaling but an agonist of Notch signaling [16], while targeting of EGFR caused CARP-1 increase and apoptosis [8]. We have previously observed increased resistance to apoptosis induced by chemotherapeutic drugs including ADR, Etoposide, CFMs, or EGFR TKI Gefitinib in cells where CARP-1 was knocked down, implicating its critical role in growth inhibition by these agents [7, 8, 11]. Given that EGFR TKIs remain frontline therapies for a large subset of NSCLCs, and emergence of resistance to TKIs continues to be a significant and unmet challenge, we investigated (a) whether IL-15 CFM compounds inhibit NSCLC cell growth and (b) the molecular mechanisms by which CFMs inhibit growth of NSCLC cells. In addition, we investigated whether CFMs will also inhibit growth of TKI-resistant NSCLC cells. To this end, we first generated and characterized laboratory models of NSCLC cells that harbor mutant EGFR and are resistant to Erlotinib, Rociletinib, or Ocimertinib. Our studies revealed that CFM Lenvatinib mesylate compound 4.16 inhibited growth of parental and also the TKI-resistant NSCLC cells when used as a single agent. CFM-4.16 synergized with B-Raf-targeting therapies (Sorafenib or Dabrafenib) and also 0.05 relative to the respective DMSO-treated controls. We next determined whether CFMs also inhibit growth of the EGFR TKI-resistant NSCLCs. We first developed and characterized NSCLC cells that were resistant to EGFR TKIs Erlotinib, Rociletinib, or Osimertinib by culturing them in the continual presence of the respective TKIs until resistance was observed. Since, Erlotinib is frequently used in clinic for treatment of the NSCLC tumors with activating mutation in the kinase domain of EGFR [4], we chose the HCC 827 NSCLC cells with EGFR exon 19 (19) mutation for generation of the Erlotinib-resistant cells. As shown in Table ?Table1,1, the GI50 doses of Erlotinib Lenvatinib mesylate for parental and resistant HCC827 cells were 0.1 M and 15 M, respectively. With growing evidence suggesting that development of resistance the TKIs Erlotinib or Gefitinib often involves activation as well as overexpression of other RTKs such as cMet or Alk, a significant subset of resistant tumors often also acquire additional, activating mutations in EGFR Lenvatinib mesylate kinase domain. These mutations include the L858R change as well as the gatekeeper T790M substitution that collectively render EGFR to become constitutively active [4]. Additional allosteric, non-ATP-competitive EGFR TKIs were recently identified and the two compounds Rociletinib and Osimertinib were tested in clinical trials with subsequent and recent FDA approval of Osimertinib for use in treatment of resistant NSCLCs. Since recent laboratory studies have reported development of resistance to Rociletinib or Osimertinib in NSCLC cells [5], we chose H1975 NSCLC cells with EGFR T790M and L858R mutations for generation of Rociletinib or Osimertinib-resistant cells. The GI50 doses for Rociletinib and Osimertinib for the parental H1975 cells were 0.18 and 0.17 M, respectively. Although the pools of the Osimertinib-resistant H1975 cells had the GI50 dose of 12 M, the GI50 doses of Rociletinib ranged from 4.5 to 8.0 M for the Rociletinib-resistant H1975 sublines. Of note is.
Briefly, the cells were washed twice in 1 PBS and trypsinized following established protocols