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Busaidy, Naifa L

Cabanillas, Maria

Dadbin, Ali

Dadu, Ramona

Fujiwara, Elena McBeath

Henderson, Ying

Hofmann, Marie-Claude

Kunnimalaiyaan, Muthusamy

Lai, Stephen

Nurieva, Roza, PhD

Stephan, Clifford

Wang, Jennifer R

Zafereo, Marc E.

English

OpenWorks @ MD Anderson

PurposeTo identify effective drug combinationstrategies targeting multiple oncogenicpathways in aggressive thyroid cancers.BackgroundTreatments with BRAF inhibitors such asdabrafenib induce significant tumor sizereduction in BRAFV600E-mutatedaggressive papillary (PTC) andanaplastic thyroid carcinoma (ATC)patients. However, these drugs areultimately ineffective due to thedevelopment of resistance, probablybecause the cells reactivate multipleoncogenic pathways used to bypass thetreatment. Therefore, novel therapeuticstrategies are still urgently needed. Weused reverse-phase protein arrays(RPPA), TCGA analysis, and RNA-Seq(GEO datasets) analyses of PTC andATC patient samples to identifycomponents of the p38/MAPK pathwayas possible targets. This study reportsthe in vitro and in vivo effects ofpharmacological inhibitors of p38/MAPKused alone or in combination with BRAFinhibitors on pre-clinical samples.Pre-Clinical Models1) ATC mouse model. We establishedTPO-CreERT2; BRAFCa/+; p53fl/flmice thatdevelop ATC tumors when treated withtamoxifen (Figures 1A +1B). Thesetumors show the same histology ashuman tumors (Figure 1C)2) Mouse cell lines. From these tumors,we established several cell lines,including MCH2.2 and PPA6 (Figure 1D)3) Human cell lines. We used thehuman cell lines MDA-T85 (PTC) andSW1736 (ATC)Conclusions• Many oncogenic molecules and pathwaysare activated by long-term culture withdabrafenib, a BRAFV600E inhibitor• Searching for downstream targets orparallel pathways through data miningrevealed overexpression/activation of thep38/MAPK14 pathway in PTC and ATCpatient samples• The p38/MAPK14 inhibitor ralimetinibsynergizes with dabrafenib, in particular inhuman PTC cells and mouse ATC cells• Ralimetinib and other p38/MAPK inhibitorsare efficient in mice bearing ATC tumorsFuture work• Test ETV5 as a marker of resistance inaggressive thyroid cancers• Test combination therapies lenvatinib andralimetinib for BRAF wild type thyroidcancers• Test the interplay between p38/MAPK14and TP53 mutations, also the role ofSTAT3 in ATCsTherapeutic Potential of Ralimetinib, a p38/MAPK14 Inhibitor, in Anaplastic Thyroid CancerMuthusamy Kunnimalaiyaan1, Ali Dadbin1, Ying Henderson2,Elena McBeath Fujiwara1, Clifford Stephan3, Roza Nurieva4, Naifa L. Busaidy1, Jennifer R Wang2, Stephen Lai2, Maria Cabanillas1, Marc Zafereo2, Ramona Dadu1, and Marie-Claude Hofmann1.1Department of Endocrine Neoplasia & HD, MDA; 2Department of Head & Neck Surgery, MDA; 3Texas A&M Institute for Biosciences and Technology, 4Department of Immunology, MDAWorkflowFigure 1: ATC mouse model and derived cell lines.A, B: Tumors develop about 3-4 months aftertamoxifen injection. C: Histology (A&E) shows typicallack of organization and high cellular de-differentiation. D: PPA6 cell line.Figure 2: Mice bearing tumors can be directly treatedwith combinations of inhibitors. Alternatively, tumorcells can be manipulated in vitro (e.g. shRNA) to testpathways, then implanted into syngeneic mice.Because the mice are immunocompetent, this alsoallows combination treatments with immunecheckpoint inhibitors.Methods and ResultsReverse Protein Array (RPPA) analysisTo test which molecules/pathways areinduced over time during BRAF inhibitorstreatment, and if dedifferentiation oftumor cells occurs, we cultured BRAF-mutated PTC and ATC cells for 8 monthswith dabrafenib as follows:Results (heatmap) indicate strong up-regulation/activation of specific pathwaysover 8 months treatment (arrows),including the MAPK/ERK1/2 (p44/42MAPK) pathway (Figure 3).Western blotsFigure 3: Part of a RPPA supervised analysis revealingpotential oncoproteins upregulated/activated over 0, 4,6, and 8 months treatment with dabrafenib in MDA-T85cells (human PTC).Figure 4: Western blots validating major pathways up-regulated over time. A: MDA-T85 human PTC cells. B:MCH2.2 mouse ATC cellsMethods and Results (cont.)The expression of ETV5, a transcriptionfactor driven by the MAPK pathway inthese cells, was not downregulated bydabrafenib over time (Figure 4).Therefore, we searched for additionalmolecules/pathways co-expressed withETV5 (TCGA and NIH GEO databases)in BRAF-mutated human PTC and ATCsamples (Figures 5 and 6):Figure 5: TCGA analysis through cBioportal showedstrong association between ETV5 and p38/MAPK14expression in 497 PTC patients.Figure 6: TCGA analysis through cBioportal showedstrong association between mutated BRAF (yellowdots) and MAP3K2 (MKK3), a kinase thatspecifically phosphorylates and activates p38MAPKin 397 samples of PTCsFigure 7: Similar to PTCs, expression of ETV5 andp38/MAPK14 is significantly higher in ATC patientsamples than in normal samples. A: ETV5expression, B: p38/MAPK14 expression. From NCBIGEO GSE65144.Methods and Results (cont.)Our data demonstrate a good synergybetween ralimetinib and dabrafenib usingcell lines in vitro (Figure 8) and in vivo(Figure 9).ABCFigure 8: High-throughput screening to identifysynergistic drug combinations. Log molarconcentrations are indicated. Green = synergy, black= additivity, red = antagonism. A: MDA-T85 humanPTC cells, B: SW1736 human ATC cells, C: MCH2.2mouse ATC cells.We next tested ralimetinib and otherinhibitors of p38/MAPK14 for possiblereduction of PTC and ATC cell growth incombination with dabrafenib.Normal ATC02004006008001000p38/MAPK14 expression valuesP = 0.0064✱✱Normal ATC02004006008001000ETV5 expression valuesP < 0.0001✱✱✱✱?p38?ralimetinibdabrafenib0100200300400500600700Day 0 Day 4 Day 12Average Tumor Volume (mm^3)Control (saline)DabrafenibRalimetinibPD169316 SB203580HarmineXanthohumolFigure 9: Growth of MCH2.2. ATC tumor cells insyngeneic mice in presence of dabrafenib or differentp38/MAPK14 inhibitors. Harmine is a TWIST1inhibitor. Xanthohumol is a NOTCH inhibitor andserves as negative control.Acknowledgements: We thank the ATCMultidisciplinary Petrick Research Funds, theIBT Drug Discovery Program, and NIH P30CA016672 MDA Core support grant.

Therapeutic Potential of Ralimetinib, a p38/MAPK14 Inhibitor, in Anaplastic Thyroid Cancer

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1028&context=leading-edge

Therapeutic Potential of Ralimetinib, a p38/MAPK14 Inhibitor, in Anaplastic Thyroid Cancer

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