c-Raf In Cells Research Articles

HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients

SummaryInhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway.

Abstract B15: The pan-BCR-ABL inhibitor ponatinib inhibits viability of gatekeeper mutant BCR-ABLT315I cells with greater potency than a nilotinib/MEK inhibitor combination

Abstract Introduction: The second generation BCR-ABL inhibitor nilotinib has recently been shown to have weak off-target activity against RAF and drive the paradoxical activation of BRAF and CRAF in cells with activated RAS. Importantly, this effect has been seen in cells expressing T315I mutant BCR-ABL, against which nilotinib is ineffective (Packer, Cancer Cell 20:715). Interestingly, nilotinib was shown to synergize with an inhibitor of MEK, a downstream effector of RAF, to inhibit the viability of cells expressing BCR-ABLT315I. Ponatinib is a pan-BCR-ABL inhibitor that potently kills cells expressing BCR-ABLT315I and is in a pivotal phase 2 trial in patients with CML. Here we examined the activity of ponatinib against RAF and compared the cellular potency of ponatinib against BCR-ABLT315I to that of a nilotinib/MEK inhibitor combination. Results: Ponatinib potently inhibited the in vitro kinase activity of ARAF, BRAF, and CRAF with IC50s of 71, 33, and 17 nM, respectively, compared to IC50s of 4390, 235, and 188 for nilotinib. In 2 RAS mutant cell lines tested, nilotinib (1 to 10 μM) induced feedback activation of RAF/MEK/ERK signaling as reported previously; however ponatinib (0.01 to 10 μM) did not. Both ponatinib and nilotinib potently inhibited viability of Ba/F3 cells expressing native BCR-ABL, with IC50s of 1.5 and 10 nM, respectively. While nilotinib has no activity against cells expressing BCR-ABLT315I, when combined with a concentration of the MEK inhibitor AZD6244 that inhibits MEK signaling (2 μM), nilotinib inhibited viability with an IC50 of 550 nM. In contrast, ponatinib potently inhibited viability of cells expressing BCR-ABLT315I with an IC50 of 6 nM. Similarly, single agent ponatinib induced apoptosis in BCR-ABLT315I cells with 100-fold greater potency than the combination of nilotinib and AZD6244. Conclusion: In contrast to nilotinib, ponatinib does not appear to induce the paradoxical activation of RAF/MEK/ERK signaling in RAS mutant cells, possibly due to its inhibition of all 3 RAF family members. Although nilotinib synergizes with a MEK inhibitor to kill BCR-ABLT315I mutant cells, this effect requires a concentration of nilotinib approximately 50-fold higher than that required to kill native BCR-ABL cells. Substantially greater potency, at clinically achievable drug concentrations, is achieved via the direct inhibition of BCR-ABLT315I by ponatinib.

Non-steroidal anti-inflammatory drugs suppress the ERK signaling pathway via block of Ras/c-Raf interaction and activation of MAP kinase phosphatases

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit cancer cell growth, induce apoptosis and decrease tumor metastasis. We have previously reported that a NSAID NS398 repressed the expression of matrix metalloproteinase-2 (MMP-2) via inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we investigate the underlying mechanism of this inhibition. In vitro kinase assay indicated that NS398 could not directly inhibit c-Raf, MEK1 and ERK enzymatic activity. We found that NS398 increased the inhibitory phosphorylation of Ser259 in c-Raf, attenuated membrane recruitment of c-Raf and inhibited Ras/c-Raf interaction to attenuate activation of this kinase. This is a general effect for NSAIDs because sulindac sulfide, aspirin and indomethacin also inhibited the binding of c-Raf to Ras. Immunofluorescent staining verified that NS398 reduced the serum-induced membrane recruitment of c-Raf in cells. However, overexpression of constitutively active c-Raf only partly reversed NS398-induced inhibition of MMP-2 expression. Interestingly, we found that NS398 up-regulated the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and MKP-3. Block of MKP activity by sodium orthovanadate also partly counteracted the inhibitory effect of NS398. Overexpression of constitutively active c-Raf and treatment of sodium orthovanadate together completely reversed the inhibition of MMP-2 by NS398. Taken together, we conclude that NS398 and other NSAIDs act via inhibition of Ras/c-Raf interaction and up-regulation of MKPs to suppress the ERK-mediated signaling.