Christina Meeks’s research while affiliated with University of Kentucky and other places

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Publications (2)


ABL1/2 are activated during acquired MEKi resistance, and drugs targeting ABL1/2 reverse intrinsic and acquired resistance, and induce apoptosis. (A) Establishment of resistant cell lines. Parental (P) cells harboring mutant NRAS were incubated with increasing concentrations of trametinib until the lines were resistant to 20 nM (-MR). (B) Cell viability assays (CellTiter Glo-CTG). Parental (P) and resistant (MR) cells were treated for 72 h (SK-MEL-30, SK-MEL-2) or 96 h (SK-MEL-147) with the indicated trametinib doses. Mean ± SEM for n = 3 independent experiments. Arrows/numbers indicate the fold difference in trametinib sensitivity between parental and resistant cell lines. (C) ABL1/2 kinase activities were indirectly assessed by Western blot analysis using an antibody that recognizes the ABL1/2 phosphorylation sites on substrates CRK/CRKL (termed pCRKL), a well-accepted read-out of ABL1/2 activities [14,15,16,17,18,19]. Quantitation for n = 3, mean ± SEM is shown. * p < 0.05 (left→right; p = 0.016, 0.028), ** p = 0.002. (D–F) Cell viability assays (CTG) using cells treated with vehicle, trametinib, nilotinib (5–6 μM), or the combination (72 h). Three doses of each drug were utilized; additional doses are shown in Figure S1. Mean ± SEM for n = 3. *** p < 0.001. Actual p-values (left→right): (D): 0.00032; (E): p < 0.0001, 0.000726; (F): 0.000101; 0.000627. (G) Clonogenic (colony) assays. Cells were treated with trametinib (Tra; 20 nM) and/or nilotinib (5 μM for SK-MEL-147; 2.5 μM for other two lines) for 7 days, washed, incubated without drugs for an additional 4 (SK-MEL-2, SK-MEL-147) or 6 (SK-MEL-30) days, and then the colonies were stained with crystal violet. Images are representative of n = 3–4. (H) Western blot analysis using attached and detached cells following treatment with nilotinib (Nilo, 2.5 μM) and/or trametinib (Tra, 20 nM) for 24 h (SK-MEL-2P, SK-MEL-2MR), 48 h (SK-MEL-30P, SK-MEL-30MR, SK-MEL-147P), or 96 h (SK-MEL-147MR). Results are representative of n = 3. cCasp-3 = cleaved caspase-3; cPARP = cleaved PARP. The uncropped blots are shown in File S1.
ABL1/2 and DDR1 are required for MEKi resistance in SK-MEL-2MR and SK-MEL-30MR. (A,B,D,G) Clonogenic assays. Plated cells were drug-treated for 7 days (A: SK-MEL-147MR; B,D,G) or 14 days (A: other cell lines) and 7 day treatment wells incubated without drugs for an additional 4 days. Drug doses are as follows: trametinib (Tra; 20nM), GNF-5 (GNF; 12.5 μM), ponatinib (pona, 100 nM), DDR-IN-1 (DDR1i, 2.5 μM), and ABL001 (A001, indicated doses. Quantitation indicates mean ± SEM for n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. Exact p-values (left→right): 0.0011, 0.037, 0.016, 0.000125, 0.0007, 0.00069 using single sample t-tests. (C) CellTiter Glo (CTG) viability assays (72 h) using resistant lines treated with specific inhibitors targeting KIT/CSF (PLX3397, KITi), DDR1 (DDR-IN-1, DDR1i), or PDGFR (CP673451, PDGFRi) −/+ trametinib (20 nM). Mean ± SEM for n = 3. (E,H) Western blots using parental or resistant cell lysates (E) or lysates from resistant cells treated with GNF-5 or ABL001 (A001, 10 μM) for 96 h (using attached and detached cells, (H)). (E) Mean ± SEM, n = 3 * p < 0.05 (0.016, 0.03; left→right). cCasp-3 = cleaved caspase-3; cPARP=cleaved PARP. (F) Cells expressing vector or activated forms of ABL1 and ABL2 (PP) were plated on tissue culture plates (−) or collagen I-coated plates (+) and treated with vehicle (−) or trametinib (+, 0.75 nM) for 72 h (top) or 18 h (bottom), followed by viability assay (CTG, top) or Western blot (bottom). Mean ± SEM, n = 3. One-way ANOVA p < 0.0001. Bonferroni’s multiple comparison tests were used to compare each treatment group to vector/no collagen-trametinib-treated cells. p-values (left→right): <0.0001, 0.001, 0.0001. (I,J) Clonogenic assays using cells from a SK-MEL-147P xenograft that developed trametinib resistance, in vivo (I) or parental cells stably expressing vectors or ABL1/2-PP, treated with trametinib (2.5 nM) for 3d, and incubated without drugs for 18d (J). Drug doses are as folows: trametinib (Tra, 20 nM), nilotinib (2.5 μM), GNF-5 (GNF, 12.5 μM), ABL001 (A001, 10 μM). Results are representative of n = 3. The uncropped blots are shown in File S1.
CRAF/ERK and DDR1 contribute to ABL1/2 activity potentiation during acquired MEKi resistance. (A,B) Phospho-CRKL expression, a reliable read-out of ABL1/2 activities [14,15,16,17,18,19], was assessed by Western blotting of lysates from parental (P) and/or resistant (MR) cells treated with vehicle or DDR1 inhibitor, DDR-IN-1 (4 μM; DDR1i), for 24 h (A) or transfected with scrambled or DDR1 siRNA targeting the 3’UTR (B). Results with a second siRNA are shown in Figure S3A. Quantitation is mean ± SEM, n = 3. (A): * p = 0.026, ** p = 0.0032. (B): * p = 0.017, ** p = 0.0092. (C,D) Phospho-CRKL expression was assessed in lysates from cells treated with trametinib (20 nM) in the absence or presence of the ERK inhibitor SCH772984 (0.1 μM) for 24 h (C) or transfected with scrambled (−), BRAF, or CRAF (5 nM) siRNA for 72 h (D). (C) Quantitation is mean ± SEM, n = 3−4. 30 MR, * p = 0.016. 147 MR, * p = 0.019. Modulation of ABL1/2 activities (pCRKL) following ARAF knockdown is shown in Figure S3B. (E,F) Mass spectrometry results for recombinant ERK2 phosphorylation of recombinant ABL1 and ABL2, in vitro. Table Inset: ERK2 phosphorylation sites in ABL1b and ABL2. Yellow highlight indicates a putative 14-3-3 binding site (14-3-3 Pred program) [17,30]. MS profiles of phosphorylated and unmodified peptide ions for GSALGTPAAEPVTPTSK on ABL1 (left) and VPVLISPTLK on ABL2 (right). Additional mass spectrometry profiles are provided in Figure S3C,D. The uncropped blots are shown in File S1.
ABL1/2 and DDR1 are required for ERK/MYC/ETS1/RSK1 reactivation during acquired resistance. (A,C,D) Western blots from cells treated with drugs or vehicle (Veh; DMSO) for 24 h (A) or 48 h (D). Drug doses are as follows: trametinib (Tra; A,C: 20 nM; D: 10 nM), nilotinib (Nilo; 2.5 μM), DDR-IN-1 (DDR1i, 4 μM-C or 2.5 μM-D).Experiments with GNF-5 (D) used lower doses of DDR1i and trametinib and a longer time (48 h) in order to increase the efficiency of GNF-5-mediated inhibition of ABL1/2. A second cell line is shown in Figure S4A,B. Graphs are mean ± SEM. (A) pERK: *** p < 0.0001, ** p = 0.008; pETS1: *** p < 0.0001, * p = 0.039. (D) *** p < 0.0001, ** p = 0.003, * p = 0.027. (B,G) RNA-seq/GSEA comparing resistant to parental cells (B) or trametinib-treated resistant lines to trametinib+nilotinib (G). (B) Only one Biocarta pathway was changed for SK-MEL-30MR. (G) Trametinib + nilotinib did not induce Biocarta pathway changes in SK-MEL-147MR. Data is shown in Figures S2 and S5 and Datasets S1,S3. (E,F) Western blots from lines transfected with DDR1 (10 nM) or ABL1/2 (20 nM) siRNAs for 72 h. Blots are representative of n = 3. A second line is shown in Figure S4C. pCRKL blots for Figure 4C,E are from the same experiment as Figure 3A,B and Figure S3A. The uncropped blots are shown in File S1.
Targeting ABL1/2 and DDR1 promotes RAF heterodimerization and degradation in trametinib-resistant cells. (A) Western blots using cells treated with vehicle (Veh), trametinib (Tra, 20 nM), and/or nilotinib (Nilo, 2.5 μM, 24 h). Mean ± SEM, n = 3–5; * p < 0.05, ** p ≤ 0.01, *** p < 0.001. Exact p-values (left→right): 0.0011, 0.0008, 0.03, 0.004, 0.006. (B) Protein stability assays. Cells were drug-treated for 24 h followed by cycloheximide treatment (CHX, 100 μM). Quantitation relative to GAPDH is shown below the blots, and quantitation for n = 3 can be found in Figure S6B–K. For SK-MEL-30MR, blots are contiguous: white bar removes an unnecessary lane. (C,D) Western blots using cells transfected for 72 h with scrambled (-), BRAF, CRAF (C), or ARAF (D) siRNA (5 nM). Blots are representative of n = 3. Some controls are the same as in Figure 3D since they are from the same replicate. (E,F) Coimmunoprecipitations (coIPs). RAF isoforms were IP’d from treated cells from (A) ((E), 24 h), or from cells treated with GNF-5 (12.5 μM) + DDR1i (2.5 μM) (F), and subjected to Western blot. To increase the efficiency of ABL1/2 inhibition by GNF-5, cells were treated for 48 h using a lower DDR1i dose. IgG is the isotype negative control IP. CoIPs for a second cell line are shown in Figure S6L. Representatives of n = 2–3 independent experiments are shown. The uncropped blots are shown in File S1.

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ABL1/2 and DDR1 Drive MEKi Resistance in NRAS-Mutant Melanomas by Stabilizing RAF/MYC/ETS1 and Promoting RAF Homodimerization
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February 2023

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66 Reads

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2 Citations

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Christina Meeks

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Simple Summary NRAS-mutant melanoma is a highly aggressive subtype with few treatment options. Although both BRAF-mutant and NRAS-mutant melanomas have activation of the MEK/ERK pathway, MEK inhibitors (MEKi) are only effective for the BRAF-mutant subtype. The aim of this study was to understand why MEKi are ineffective in NRAS-mutant melanomas with the long-term goal of identifying new treatment regimens. Here, we show that ABL and DDR kinases are critically important for MEKi resistance because they cooperate to promote the stability of key proteins involved in driving melanoma growth and survival. FDA-approved drugs that inhibit ABL1/2 and DDR1 have been used for decades to treat leukemia. We showed that one such inhibitor prevents MEKi resistance from developing in a NRAS-mutant melanoma animal model. Thus, the data in this study provide the rationale for testing the use of drugs targeting ABL1/2 and DDR1 in combination with MEKi for patients with NRAS-mutant melanomas who have failed to respond to immunotherapy. Abstract Melanomas harboring NRAS mutations are a particularly aggressive and deadly subtype. If patients cannot tolerate or the melanomas are insensitive to immune checkpoint blockade, there are no effective 2nd-line treatment options. Drugs targeting the RAF/MEK/ERK pathway, which are used for BRAF-mutant melanomas, do little to increase progression-free survival (PFS). Here, using both loss-of-function and gain-of-function approaches, we show that ABL1/2 and DDR1 are critical nodes during NRAS-mutant melanoma intrinsic and acquired MEK inhibitor (MEKi) resistance. In some acquired resistance cells, ABL1/2 and DDR1 cooperate to stabilize RAF proteins, activate ERK cytoplasmic and nuclear signaling, repress p27/KIP1 expression, and drive RAF homodimerization. In contrast, other acquired resistance cells depend solely on ABL1/2 for their survival, and are sensitive to highly specific allosteric ABL1/2 inhibitors, which prevent β-catenin nuclear localization and destabilize MYC and ETS1 in an ERK-independent manner. Significantly, targeting ABL1/2 and DDR1 with an FDA-approved anti-leukemic drug, reverses intrinsic MEKi resistance, delays acquisition of acquired resistance, and doubles the survival time in a NRAS-mutant mouse model. These data indicate that repurposing FDA-approved drugs targeting ABL1/2 and DDR1 may be a novel and effective strategy for treating patients with treatment-refractory NRAS-driven melanomas.

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Combating acquired resistance to MAPK inhibitors in melanoma by targeting Abl1/2-mediated reactivation of MEK/ERK/MYC signaling

October 2020

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225 Reads

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29 Citations

Metastatic melanoma remains an incurable disease for many patients due to the limited success of targeted and immunotherapies. BRAF and MEK inhibitors reduce metastatic burden for patients with melanomas harboring BRAF mutations; however, most eventually relapse due to acquired resistance. Here, we demonstrate that ABL1/2 kinase activities and/or expression are potentiated in cell lines and patient samples following resistance, and ABL1/2 drive BRAF and BRAF/MEK inhibitor resistance by inducing reactivation of MEK/ERK/MYC signaling. Silencing/inhibiting ABL1/2 blocks pathway reactivation, and resensitizes resistant cells to BRAF/MEK inhibitors, whereas expression of constitutively active ABL1/2 is sufficient to promote resistance. Significantly, nilotinib (2nd generation ABL1/2 inhibitor) reverses resistance, in vivo, causing prolonged regression of resistant tumors, and also, prevents BRAFi/MEKi resistance from developing in the first place. These data indicate that repurposing the FDA-approved leukemia drug, nilotinib, may be effective for prolonging survival for patients harboring BRAF-mutant melanomas.

Citations (2)


... A more recently identified negative regulators of oligodendrocyte differentiation is the MAPK/ERK pathway [27,[45][46][47]. It was previously demonstrated that Ddr1 could modulate the activity of the AKT/ERK pathway and plays an important role in the migration and adhesion of cancer cells [30,48,49]. The Western blot analysis confirmed that inhibition of Ddr1 upregulates the ratio of p-ERK to total ERK without altering the AKT signaling pathway (Figure 7). ...

Reference:

Evidence That DDR1 Promotes Oligodendrocyte Differentiation during Development and Myelin Repair after Injury
ABL1/2 and DDR1 Drive MEKi Resistance in NRAS-Mutant Melanomas by Stabilizing RAF/MYC/ETS1 and Promoting RAF Homodimerization

... In the literature, the molecular anti-tumoral effects of Encorafenib have been previously determined in different MM cell lines 9-11 . The activated pathways and related molecular mechanisms for Vemurafenib and Dabrafenib resistance have also been well-defined by establishing various drug-resistant MM cells [12][13][14][15][16] . Moreover, although a previous study investigated Encorafenib + Binimetinib resistance mechanisms by performing RNA-Seq and protein array analysis in MM cell lines 17 , there is no study in the literature yet in which the effect of Encorafenib resistance mechanisms and molecular alterations in the pathways activated during the acquired Encorafenib resistance. ...

Combating acquired resistance to MAPK inhibitors in melanoma by targeting Abl1/2-mediated reactivation of MEK/ERK/MYC signaling