RAF Inhibition In Melanoma Research Articles

Map3k8/COT1/Tpl2 Drives Resistance to Bcr-Abl1 Inhibition through Activation of MEK-ERK and NF-Kb Pathways

The introduction of tyrosine kinase inhibitors (TKI) has transformed chronic myeloid leukemia (CML) into a manageable, chronic disease with long-term survival exceeding 85%, but discontinuation of TKI in patients who achieve deep molecular remissions results in a 50% relapse rate. The relapses are believed to be caused by Bcr-Abl1-expressing hematopoietic stem cells (CML stem cells) which are independent of the Bcr-Abl1 kinase activity for their survival. Signaling pathways, including PI3K/AKT, Wnt/β-catenin, JAK/STAT, and Hedgehog/SMO, are crucial for CML stem cell self-renewal and survival in the presence of TKI. Eradication of these cells poses the greatest current challenge.We previously described a Bcr-Abl1-independent model of resistance to imatinib mesylate (IM), in which K562 cells cultured in high concentration of IM displayed reduced Bcr-Abl1 protein and activity levels (Chorzalska et al., Leukemia 2014). Quantitative phosphoproteomic analysis of these IM-resistant cells (IMRC) identified 2,593 phosphopeptides showing significant differences in abundancies in comparison to IM-sensitive cells. Functional analysis of identified phosphopeptides pointed to MEK-ERK pathway as a highly dysregulated cascade in IMRC (Chorzalska et al., Stem Cell Dev, 2017). Within the list of identified phosphopeptides, we assessed the abundancy of 222 MEK-ERK pathway-specific core interactor peptides (as listed in MEK-ERK superpathway in Pathcards), and identified the Map3k8-derived phosphopeptide as the most abundant one in IMRC (Fig. 1A). Map3k8, also known as Tumor Progression Locus (Tpl2) or Cancer Osaka Thyroid (COT1), links the MEK-ERK and NF-κB signaling and drives resistance to Raf inhibition in melanoma, but its role in chemoresistance of CML stem cells has not been explored.Using RT-PCR assays in CML CD34+ cells cultured with IM for 7 days, we found a 2-fold increase in MAP3K8 transcriptsand a 1.7-fold increase in transcripts of its binding partner NFKB1 (NF-κB1; Fig. 1B). IMRC also showed increased expression of Map3k8 and NF-κB1 protein levels, and increased activity of the NF-κB signaling components: NF-κB inhibitor - IκB and IκB kinase - IKKα/β (Fig. 1C). Immunoblotting evaluation of the Map3k8-dependent effect on MEK-ERK signaling indicated no significant difference between IMRC and control cells in the activity of Raf module, with exception of C-Raf Ser289/296/301 inhibitory sites associated with a MEK-ERK negative feedback loop (Fig. 1D). Evaluation of the downstream effectors of Raf showed no difference in MEK1 and decrease in MEK2 abundance, and enhanced phosphorylation of MEK1/2. IMRC also showed higher ERK1 and lower ERK2 abundance, and no significant change in the stoichiometry of ERK1/ERK2 phosphorylation at Thr202/Tyr204 (Fig. 1D). To identify other upstream mediators of MEK phosphorylation, we evaluated the activity of Src Family Kinases (SFKs) and found a marked increase in phospho-SFKs in IMRC (Fig. 1E).Considering the observed upregulation of SFKs, MEK and NF-κB signaling in Map3k8-overexpressing IMRC, we investigated apoptotic effects of the SFKs inhibitor dasatinib, the MEK inhibitor U0126, and the IKK inhibitor PS-1145, individually and in combination. The SFKs and IKK inhibitors, as single agents or combined, induced only a modest (35%) increase in apoptosis. Conversely, the combination of SFKs+MEK inhibitors decreased survival of IMRC by nearly 50%, whereas the triple SFKs+MEK+IKK inhibition killed 65% of IMRC within 48h of incubation (Fig. 1F). In addition, the combination of three inhibitors SFKs+MEK+IKK significantly decreased the phosphorylation of MEK, ERK, SFKs and NF-κB, surpassing the effect of a dual SFKs+MEK or SFKs+IKK inhibition (Fig. 1G). The effect of SFKs, MEK and IKK inhibitors on the capacity of CML CD34+ cells to form colonies in methylcellulose assays was also evaluated. The dual SFKs+MEK inhibition reduced the colony number by 70%, while the triple SFKs+MEK+IKK inhibition decreased it by about 80% (Fig. 1H). Finally, CML CD34+ cells cultured for 7 days in the presence of SFKs+MEK+IKK inhibitors showed significant reduction of MAP3K8 transcript levels (Fig. 1I).In conclusion, combined inhibition of SFKs, MEK and NF-κB attenuates the survival of IM- resistant CML cell line and CML stem/progenitor cells, and a combination of relevant inhibitors may offer a new therapeutic option for overcoming the TKIs resistance in CML patients. [Display omitted] DisclosuresReagan:Teva: Membership on an entity's Board of Directors or advisory committees.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The genetic landscape of clinical resistance to RAF inhibition in melanoma.

11009 Background: Although single-agent RAF inhibition has proved effective in metastatic BRAFV600-mutant melanoma, most patients relapse and some are intrinsically resistant. While several genetic resistance effectors have been identified, a comprehensive assessment of the genetic resistance spectrum in a large patient cohort may further inform resistance patterns and treatment strategies. Methods: Pre-treatment and post-relapse biopsies were obtained from BRAFV600melanoma patients treated with vemurafenib or dabrafenib. Whole exome sequencing of tumor and normal samples was performed to identify exome-wide mutations, insertion/deletions, and chromosomal copy number alterations. Since somatic allelic fraction comparisons across treatment time points must account for variable stromal admixture between biopsies, novel algorithms were applied to impute purity and tumor cell ploidy, thus determining the “cancer cell fraction” (CCF) of each alteration. Those with enriched CCF in post-relapse samples were nominated as potential resistance effectors. Results: 30 patients (towards a goal of 77) were sequenced to a mean depth of 145-fold coverage. In acquired resistance patients (on drug ≥ 12 weeks, n=18), NRAS Q61R (28%) and BRAF amplifications (6%) were observed. The remaining patients had multiple clonally enriched alterations in the post-relapse tumors that may drive resistance, including MEK1 mutations at residues previously noted in an in vitro mutagenesis screen for vemurafenib resistance. In the intrinsic resistance cohort (on drug < 12 weeks, n=12), multiple putative resistance mutations not previously observed in the setting of acquired resistance were identified. Conclusions: In addition to reported genetic resistance mechanisms, we identified new MEK1 mutations in resistant samples. For the majority of patients (67%) without events in these genes, computational algorithms identified new candidate drivers of RAF inhibitor resistance. Alterations that predict intrinsic or acquired resistance may also inform new approaches to melanoma therapy. Future studies incorporating longitudinal biopsies and genetic profiling will underpin a robust assessment of clinical resistance in melanoma and other cancers.

NF1 drives resistance to RAF inhibition in melanoma

NF1 drives resistance to RAF inhibition in melanoma

Resistant mechanisms to BRAF inhibitor PLX4032 in melanoma

Evaluation of: Wagle N, Emery C, Berger MF et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J. Clin. Oncol. DOI: 10.1200/JCO.2010.33.2312 (Epub ahead of print) (2011).Melanoma is a deadly skin disease well known for resistance to chemotherapies and immunomodulating therapies. PLX4032 is a selective BRAF inhibitor and, in clinical trials, has consistently induced tumor responses in the majority of patients with BRAF V600E mutant melanoma. However, duration of response has been limited due to the development of acquired resistance. This article evaluates a recently published investigation evaluating the resistant mechanisms to BRAF therapy using tumor genomic profiling of isolated DNA from responsive and acquired resistant melanoma tissue in a patient treated with the BRAF inhibitor PLX4032.

Abstract 3933: Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling

Abstract Although targeted anticancer agents often result in dramatic responses, tumors invariably become resistant to these agents. A detailed understanding of the mechanisms by which tumors acquire resistance to such inhibitors should speed the development of more durable treatment strategies. The promise of RAF inhibition in metastatic melanoma is exemplary in this regard. Mutations in the BRAF oncogene have been detected in more than 50% of metastatic melanomas, and inhibition of mutant BRAF has shown high response rates in early clinical trials of patients with melanoma. However, resistance to RAF inhibition invariably develops. Mechanisms of de novo and acquired resistance to RAF inhibition remain poorly understood. Moreover, the clinical application of genomic approaches that might be capable of diagnosing salient resistance mechanisms remains underdeveloped. Here, we describe an approach to characterize genetic mechanisms of resistance through systematic tumor mutation profiling. We performed massively parallel sequencing of 138 cancer genes in a tumor specimen from a melanoma patient who developed resistance to PLX4032 after a dramatic initial response. The resulting profile identified a novel mutation in the downstream kinase MEK1 that was absent in the corresponding pre-treatment tumor. This MEK1 mutation was shown to increase kinase activity and confer robust resistance to both RAF and MEK inhibition in vitro. Our results provide an instructive framework both for assessing mechanisms of acquired resistance to kinase inhibition and deploying new technologies for elaboration of resistance mechanisms in a manner that may accelerate personalized cancer medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3933. doi:10.1158/1538-7445.AM2011-3933