Apoptosis Induced by Cytoskeletal Disruption Requires Distinct Domains of MEKK1

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD

Abstract 2595: Efficacy of cetuximab and mutant selective EGFR inhibitor WZ4002 in EGFR T790M and non-T790M models of erlotinib resistant non-small cell lung cancer

Abstract Background: EGFR kinase inhibitors, including erlotinib, are effective treatments for patients with EGFR mutant lung cancer. However, resistance inevitably develops, most commonly (60%) mediated by an EGFR secondary mutation EGFR T790M. Mutant selective EGFR inhibitors (WZ4002, AZD9291, CO-1686) are effective in preclinical models and clinically (AZD9291 &amp; CO-1686) in EGFR T790M patients (response rates (RR) &amp;gt; 50%) but are less effective in patients with non-T790M mediated drug resistance (AZD9291 RR ∼10%). Combination of afatinib and cetuximab is effective in both T790M (32% RR) and non-T790M (25% RR) mediated resistance but is associated with significant toxicity. We evaluated whether cetuximab could add to the efficacy of a mutant selective EGFR inhibitor and whether the combination was also effective in a model of non-T790M mediated resistance. Methods: Mice harboring xenografts from PC9GR (EGFR Del 19/T790M), H1975 (EGFR L858R/T790M), HCC827GR (EGFR Del 19/MET amplification) cells or genetically engineered mouse models (GEMMS) expressing EGFR L858R/T790M were treated with vehicle, WZ4002 alone, cetuximab alone or the combination. We compared the efficacy of the treatments and evaluated for tumor cures (defined as tumor disappearance and lack of regrowth following 6 weeks of drug withdrawal). In addition, we evaluated the impact of the treatments on EGFR signaling and apoptosis. Results: In the EGFR T790M xenograft models (PC9GR and H1975), the combination of WZ4002 and cetuximab was more effective than either single agent. Only the combination led to tumor cures (5/15 in PC9GR and 3/18 in H1975) while none were observed with the single agents. In the HCC827GR xenografts, the combination of WZ4002/cetuximab was more effective than the single agents but did not lead to any cures. Evaluation of the tumors from treated mice revealed that the efficacy of the combination correlates with effective degradation of EGFR and MET (in HCC827GR). In the L858R/T790M GEMMs, the combination led to significantly greater tumor shrinkage compared to single agents. Evaluation of treated tumors revealed enhanced apoptosis in the combination treated mice. Conclusions: The addition of cetuximab enhances the efficacy of the mutant selective EGFR inhibitor WZ4002 in vivo by increasing the single agent efficacy in both EGFR T790M and non-T790M models. Clinical trials of mutant selective EGFR inhibitors and cetuximab are warranted. Citation Format: Erin M. Tricker, Chunxiao Xu, Kwok-Kin Wong, Pasi A. Janne. Efficacy of cetuximab and mutant selective EGFR inhibitor WZ4002 in EGFR T790M and non-T790M models of erlotinib resistant non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2595. doi:10.1158/1538-7445.AM2015-2595</jats:p

Abstract 1832: Combined EGFR and MEK inhibition prevents the emergence of drug resistance in EGFR mutant non-small cell lung cancer (NSCLC)

Abstract Background: EGFR inhibitors are effective clinical therapies for EGFR mutant NSCLC, but efficacy is limited by the development of acquired drug resistance. The mechanisms of resistance include secondary mutations in EGFR (T790M) and activation of compensatory signaling pathways (MET, IGFR and AXL). Mutant selective EGFR inhibitors (WZ4002, CO-1686 and AZD9291) are effective in preclinical and clinical models of EGFR T790M. Our lab has demonstrated that reactivation of ERK signaling is a mechanism of resistance to WZ4002 (WZ) which could be reversed or prevented by using a combination of WZ and MEK inhibitors. Given the broad array of possible EGFR inhibitor resistance mechanisms, we evaluated whether WZ combined with the MEK inhibitor trametinib (TRA) can prevent the emergence of resistance in vitro and in vivo. Methods and Results: We developed a plate based resistance assay in which 350 cells/well are plated in 96 well plates and treated weekly. This system allows for evaluation of drug efficacy over weeks of treatment and can be used to measure both time to development and frequency of drug resistance. Given that ERK reactivation can cause resistance to WZ, we evaluated WZ, TRA or the combination thereof (WZ/TRA) using PC9 cells. Single agents led to 100% resistance in 2-3 weeks, versus 5% with WZ/TRA in 24 weeks. We then tested WZ/TRA in 5 additional EGFR TKI sensitive cell lines, including several known to develop acquired resistance to gefitinib (HCC827, MET amplification; HCC4006, EMT or FGFR activation; HCC2279; FGFR activation; H3255, T790M; HCC2935). In all cases, WZ/TRA significantly reduced the emergence of drug resistant clones compared to each single agent. In models with established drug resistance mechanisms, WZ/TRA was effective in 3/3 models with T790M mutations, but not (0/5) in models with established non-T790M resistance mechanisms. In models where WZ/TRA was effective, the combination led to increased apoptosis compared to single agents and effective inhibition of ERK signaling. We also evaluated the effectiveness of the WZ/TRA combination in vivo using EGFR L858R/T790M genetically engineered mouse model and PC9GR4 (T790M+) xenografts. Combination WZ/TRA treatment prevented tumor outgrowth for 24 weeks in 5 EGFR L858R/T790M GEMM mice whereas individual treatments did not. Additionally, we found that WZ/TRA combination treatment, but not single agents, could cure 7/15 xenograft tumors as assessed by lack of tumor regrowth after treatment cessation. Conclusion: Our studies suggest that the combination of a mutant selective EGFR inhibitor and a MEK inhibitor can prevent the emergence of both T790M and non-T790M mediated drug resistance mechanisms. This strategy is more effective at preventing (6/6) than treating (3/8) cancers with established drug resistance mechanisms and should be evaluated in clinical trials in EGFR TKI naïve EGFR mutant NSCLC patients. Citation Format: Erin M. Tricker, Chunxiao Xu, Dalia Ercan, Atsuko Ogino, Kwok-kin Wong, Pasi Janne. Combined EGFR and MEK inhibition prevents the emergence of drug resistance in EGFR mutant non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1832. doi:10.1158/1538-7445.AM2014-1832</jats:p

Mechanocatalytic hydrogenolysis of benzyl phenyl ether over supported nickel catalysts

Mechanocatalytic hydrogenolysis of benzyl phenyl ether, a model lignin ether, is performed under solvent-free conditions in a ball mill.</jats:p

MEKK1^mutP is not phosphorylated in the activation loop after vinblastine treatment.

<p><i>MEKK1^−/−</i>, MEKK1^Wt, MEKK1^mutP or MEKK1^mutK (left panel) and MEKK1^delU, MEKK1^mutC (right panel) cell lines were treated with 1 µM vinblastine for 2 hours. Cell lysates were immunoprecipitated with αHA-agarose, run on SDS-PAGE gel and probed with αphospho-MEKK1 followed by detection of total MEKK1 with αMEKK1 C-22.</p

The PHD and kinase domains are essential for MEKK1-dependent JNK activation.

<p><i>A</i>. Expression of mutations in relation to endogenous MEKK1. HA-His-tagged pBabeMEKK1 was introduced into <i>MEKK1^−/−</i> (−/−) DT40 cells by retroviral infection (reconstituted cells are referred to here as M-^Wt and MEKK1^Wt in the text), selected with puromycin and protein levels were assessed by western blot probed with αMEKK1 (left panel). The size difference between His-HA-tagged and endogenous MEKK1 is indicated. Each mutation was introduced into <i>MEKK1</i>^−/− cells and expression was compared (right panel). Reconstituted cell lines are referred to as M-^mutation within each figure. <i>B</i>. The PHD and kinase domain are required for c-Jun phosphorylation after vinblastine stimulation. Reconstituted cell lines were stimulated with 1 µM vinblastine (Vin) for four hours and lysed in modified RIPA. Phosphorylated c-Jun, total c-Jun and β-tubulin were assessed by western blot. <i>C</i>. There is a decreased amount of c-Jun phosphorylation in the <i>MEKK1</i>^−/−, PHD and kinase domain mutant cell lines after cytochalasin D treatment. Phosphorylation of c-Jun and total c-Jun levels of each reconstituted cell line was assessed by western blot after four hours of 2 µg/ml cytochalasin D (Cy) treatment. <i>D</i>. There is a decreased amount of c-Jun phosphorylation in the <i>MEKK1^−/−</i>, PHD and kinase domain mutant cell lines after okadaic acid treatment. Phosphorylation of c-Jun and total c-Jun levels of each reconstituted cell line was assessed by western blot after four hours of 90 nM okadaic acid (OA) treatment.</p

The PHD and kinase domains of MEKK1 are not essential for maximal ERK activation.

<p><i>A</i>. ERK is not activated or degraded after vinblastine treatment. Wild type, knockout and MEKK1-reconstituted <i>MEKK1^−/−</i> cells were treated with 1 µM vinblastine for four hours and total ERK or β-tubulin were assessed by western blot. <i>B</i>. Activation of ERK by cytochalasin treatment is not MEKK1-dependent. Cell lines were treated with 2 µg/ml cytochalasin D for two hours and phosphorylated and total ERK were evaluated by western blot. <i>C</i>. Activation of ERK by okadaic acid treatment is not MEKK1-dependent. Phosphorylation of ERK and total ERK were determined by western blot after treatment with 90 nM okadaic acid for four hours. β-tubulin was used as a loading control.</p

Identifying the post-translational modifications of the MEKK1 protein.

<p><i>A</i>. The PHD mutant can be phosphorylated. Vector, wild type flag-MEKK1 and flag-PHD mutant (mutP) were transfected into 293T cells and immunoprecipitated with αflag M2 conjugated beads. Membranes were probed with total or phospho-MEKK1. <i>B</i>. CIP treatment does not affect basal MEKK1 modification, but does shift the molecular weight of vinblastine treated (phosphorylated) endogenous MEKK1. DT40 cells were treated with vinblastine for six hours and lysates were immunoprecipitated with αMEKK1. Half of each lysate was treated with calf alkaline phosphatase and all were incubated at 37°, run on SDS-PAGE gel and membranes were probed with αMEKK1. <i>C</i>. Inhibition of de-ubiquitinating enzymes via N-ethylmaleimide (NEM) stabilizes the higher molecular weight form of MEKK1. Wild type DT40 cells were lysed in modified RIPA. Lysates were treated with or without 20 mM NEM for 30 minutes at room temperature. Lysates were run on SDS-PAGE and probed with α MEKK1. <i>D</i>. MEKK1 is ubiquitinated whereas the PHD mutant is not. 293T cells were transiently transfected with vector, flag-MEKK1 or mutP. Cell lysates were immunoprecipitated with flag-conjugated beads, run on an SDS-PAGE gel and probed with αubiquitin or αMEKK1.</p

Characterization of MAPK activation in wild type and MEKK1-deficient cell lines.

<p><i>A</i>. Phosphorylation of c-Jun phosphorylation is defective in the <i>MEKK1</i>^−/− chicken DT40 cells in response to cytoskeletal disruption. Wild type (Wt) and <i>MEKK1</i>^−/− DT40 cells were left untreated (U) or stimulated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy), and 90 nM okadaic acid (OA) for four hours, lysed in modified RIPA buffer and run on an SDS-PAGE gel to compare wild type and knockout cell lines. Western blot membranes were probed for phosphorylated and total c-Jun, ERK and p38. <i>B</i>. Phosphorylation of c-Jun is defective in <i>MEKK1</i>^−/− murine pre-B cells in response to cytoskeletal disruption. Wild type (Wt) and <i>MEKK1</i>^−/− pre-B murine cells were stimulated with 1 µM vinblastine, 2 µg/ml cytochalasin D, and 90 nM okadaic acid for four hours, lysed in modified RIPA buffer and run on an SDS-PAGE gel. Membranes were probed for phosphorylated and total c-Jun, ERK and p38. β-tubulin is used as loading control.</p

The PHD and kinase domains are required for vinblastine induced apoptosis.

<p><i>A</i>. <i>MEKK1^−/−</i> DT40 cells reconstituted with MEKK1 or mutations thereof all initiate apoptosis in response to etoposide. Each reconstituted cell line was treated with 25 µM etoposide (Et) for four hours and subjected to DNA laddering assay. <i>B</i>. <i>MEKK1^−/−</i> cell lines reconstituted with MEKK1 containing the PHD or kinase mutations do not result in DNA fragmentation after vinblastine treatment. Each reconstituted cell line was treated with 1 µM vinblastine (Vin) for six hours and subjected to a DNA laddering assay. <i>C</i>. There is a defective the DNA laddering response in the PHD or kinase domain mutant cell lines after cytochalasin D treatment. Reconstituted cell lines were treated with 2 µg/ml cytochalasin D (Cy) for six hours and subjected to DNA laddering assays. <i>D</i>. There is a defective the DNA laddering response in the PHD or kinase domain mutant cell lines after okadaic acid treatment. Reconstituted cell lines were treated with 90 nM okadaic acid (OA) for six hours and subjected to DNA laddering assays.</p