JUN dependency in distinct early and late BRAF inhibition adaptation states of melanoma.

A prominent mechanism of acquired resistance to BRAF inhibitors in BRAF (V600) -mutant melanoma is associated with the upregulation of receptor tyrosine kinases. Evidences suggested that this resistance mechanism is part of a more complex cellular adaptation process. Using an integrative strategy, we found this mechanism to invoke extensive transcriptomic, (phospho-) proteomic and phenotypic alterations that accompany a cellular transition to a de-differentiated, mesenchymal and invasive state. Even short-term BRAF-inhibitor exposure leads to an early adaptive, differentiation state change-characterized by a slow-cycling, persistent state. The early persistent state is distinct from the late proliferative, resistant state. However, both differentiation states share common signaling alterations including JUN upregulation. Motivated by the similarities, we found that co-targeting of BRAF and JUN is synergistic in killing fully resistant cells; and when used up-front, co-targeting substantially impairs the formation of the persistent subpopulation. We confirmed that JUN upregulation is a common response to BRAF inhibitor treatment in clinically treated patient tumors. Our findings demonstrate that events shared between early- and late-adaptation states provide candidate up-front co-treatment targets

Exploiting Drug Addiction Mechanisms to Select against MAPKi-Resistant Melanoma.

Melanoma resistant to MAPK inhibitors (MAPKi) displays loss of fitness upon experimental MAPKi withdrawal and, clinically, may be resensitized to MAPKi therapy after a drug holiday. Here, we uncovered and therapeutically exploited the mechanisms of MAPKi addiction in MAPKi-resistant BRAFMUT or NRASMUT melanoma. MAPKi-addiction phenotypes evident upon drug withdrawal spanned transient cell-cycle slowdown to cell-death responses, the latter of which required a robust phosphorylated ERK (pERK) rebound. Generally, drug withdrawal-induced pERK rebound upregulated p38-FRA1-JUNB-CDKN1A and downregulated proliferation, but only a robust pERK rebound resulted in DNA damage and parthanatos-related cell death. Importantly, pharmacologically impairing DNA damage repair during MAPKi withdrawal augmented MAPKi addiction across the board by converting a cell-cycle deceleration to a caspase-dependent cell-death response or by furthering parthanatos-related cell death. Specifically in MEKi-resistant NRASMUT or atypical BRAFMUT melanoma, treatment with a type I RAF inhibitor intensified pERK rebound elicited by MEKi withdrawal, thereby promoting a cell death-predominant MAPKi-addiction phenotype. Thus, MAPKi discontinuation upon disease progression should be coupled with specific strategies that augment MAPKi addiction.Significance: Discontinuing targeted therapy may select against drug-resistant tumor clones, but drug-addiction mechanisms are ill-defined. Using melanoma resistant to but withdrawn from MAPKi, we defined a synthetic lethality between supraphysiologic levels of pERK and DNA damage. Actively promoting this synthetic lethality could rationalize sequential/rotational regimens that address evolving vulnerabilities. Cancer Discov; 8(1); 74-93. ©2017 AACR.See related commentary by Stern, p. 20This article is highlighted in the In This Issue feature, p. 1

Exploiting Drug Addiction Mechanisms to Select against MAPKi-Resistant Melanoma.

Melanoma resistant to MAPK inhibitors (MAPKi) displays loss of fitness upon experimental MAPKi withdrawal and, clinically, may be resensitized to MAPKi therapy after a drug holiday. Here, we uncovered and therapeutically exploited the mechanisms of MAPKi addiction in MAPKi-resistant BRAFMUT or NRASMUT melanoma. MAPKi-addiction phenotypes evident upon drug withdrawal spanned transient cell-cycle slowdown to cell-death responses, the latter of which required a robust phosphorylated ERK (pERK) rebound. Generally, drug withdrawal-induced pERK rebound upregulated p38-FRA1-JUNB-CDKN1A and downregulated proliferation, but only a robust pERK rebound resulted in DNA damage and parthanatos-related cell death. Importantly, pharmacologically impairing DNA damage repair during MAPKi withdrawal augmented MAPKi addiction across the board by converting a cell-cycle deceleration to a caspase-dependent cell-death response or by furthering parthanatos-related cell death. Specifically in MEKi-resistant NRASMUT or atypical BRAFMUT melanoma, treatment with a type I RAF inhibitor intensified pERK rebound elicited by MEKi withdrawal, thereby promoting a cell death-predominant MAPKi-addiction phenotype. Thus, MAPKi discontinuation upon disease progression should be coupled with specific strategies that augment MAPKi addiction.Significance: Discontinuing targeted therapy may select against drug-resistant tumor clones, but drug-addiction mechanisms are ill-defined. Using melanoma resistant to but withdrawn from MAPKi, we defined a synthetic lethality between supraphysiologic levels of pERK and DNA damage. Actively promoting this synthetic lethality could rationalize sequential/rotational regimens that address evolving vulnerabilities. Cancer Discov; 8(1); 74-93. ©2017 AACR.See related commentary by Stern, p. 20This article is highlighted in the In This Issue feature, p. 1

Imatinib mesylate inhibits the PDGF-BB induced signalling pathways.

<p>Human, mouse and rat osteosarcoma were treated with 50/mL of PDGF-BB for 5 minutes in the presence of the absence of 25 µM of imatinib mesylate. PDGFRα, PDGFRβ, Akt, ERK1/2 phoshorylations were analyzed by Western blot compared to the levels of total forms of proteins and the levels of actin.</p

Imatinib mesylate inhibits AKT/mTOR signaling pathway in osteosarcoma cells and activates ERK1/2 phosphorylation: PDGFRα, a key target of osteosarcoma cells.

<p>Human, mouse and rat osteosarcoma were treated with various doses of imatinib mesylate to analyse the effects of the drug on intra-cellular signaling pathways. (<b>A</b>) Imatinib mesylate inhibits mTOR and Akt phosphorylation in human HOS and mouse MOS-J cells. RAD001 named everolimus (RAD), a mTOR inhibitor was used as a positive control. (<b>B</b>) Human Phospho-receptor tyrosine kinase array Kit was used to identify the molecular targets of imatinib mesylate. (C) Expression of PDGFRα and PDGFRβ analyzed in human, mouse and rat osteosarcoma cells by semi-quantitative RT-PCR.</p

Imatinib mesylate inhibits in a dose dependent manner the osteosarcoma cells proliferation.

<p>Human (HOS, MG63) (<b>A</b>)<b>,</b> mouse <b>(</b>POS-1, MOS-J<b>)</b> (<b>B</b>) and rat (OSRGA) (<b>C</b>) osteosarcoma cell lines were treated by increasing concentration of imatinib mesylate (0.1–40 µM) for 72 hours. The number of viable cells was then determined using an XTT assay. (<b>D</b>) Table summarizing the IC50 and IC90 of each cell lines studied. Graphs represent the average values of three independent experiments performed in triplicate.</p

Imatinib mesylate inhibits osteosarcoma development in “preventive” and “curative” therapeutic context.

<p>Mice bearing undifferentiated POS-1 (<b>A, B</b>) or mixed osteoblastic/osteolytic MOS-J (<b>C–F</b>) osteosarcoma tumours (n = 8 per group) were assigned as control (vehicle), or imatinib mesylate (25, 50 or 100 mg/kg, daily oral administration). The treatment started 1 day after tumour cell inoculation (« preventive » treatment, <b>A–D</b>) or treatment started when tumours are palpable (7–10 days) named “curative treatment” (<b>E, F</b>). Evolution of tumour volumes (mm³) (A, C, E); follow-up of tumour progressions (B, D, F). * P<0.05; ** P<0.01; *** P<0.001.</p

Imatinib mesylate increases osteosarcoma cell death.

<p>A kinetic of human (MG63), mouse (MOS-J) and rat (OSRGA) osteosarcoma cell death was analyzed by time-lapse microscopy in the presence or the absence of 25 µM, 20 µM or 10 µM imatinib mesylate respectively. The number of cell death was manually scored every 10 minutes until 72 hours.</p