BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.

BRAF inhibitors are active in BRAF mutant melanoma patients, but the majority of patients will eventually develop resistance or present intrinsic resistance and so will not respond to BRAF inhibitors, despite the presence of a BRAF mutation. Here, we describe pan-RAF inhibitors that also target SRC and that are active in tumors from patients who developed resistance to BRAF-selective inhibitors and a BRAF plus MEK inhibitor combination. These compounds, therefore, provide vital second-line targeted therapies for relapsed patients, and a compound from the series is being developed to enter clinical trials.

To overcome both resistance and paradoxical activation of the MEK/ERK pathway, strategies to achieve increased inhibition of the pathway by combined targeting of BRAF and MEK have been tested. The combination of dabrafenib, a BRAF inhibitor, with trametinib, a MEK inhibitor, was recently approved by the U.S. Food and Drug Administration for treating patients with mutant BRAF melanomas, based on phase II clinical trial data that show that the combination achieved higher response rates, longer median progression-free survival, and less cutaneous toxicity than dabrafenib alone (). However, despite these improved responses, patients on this drug combination still develop resistance, and most patients relapse after ∼9 months of treatment; furthermore, a recent study reported that, in these patients, resistance can be mediated by acquired mutations in MEK2 (). Independent of the mechanisms of resistance, there is an urgent need for second-line treatments for BRAF mutant melanoma patients who develop resistance to BRAF inhibitor mono- and combination therapies.

In addition to resistance, BRAF inhibitors mediate a curious paradox. Although they inhibit MEK/ERK signaling in BRAF mutant cells, they activate MEK/ERK signaling in RAS mutant cells. This is because, in the presence of oncogenic RAS, BRAF inhibitors drive the formation of BRAF-CRAF hetero- and homodimers containing one partner that is drug bound and one partner that is drug-free. The drug-bound partner drives activation of the drug-free partner through scaffolding or conformational functions, activating CRAF and, consequently, stimulating MEK and ERK hyperactivation (). In some contexts, paradoxical activation of the pathway can stimulate tumor growth and progression.

Many mechanisms of resistance to BRAF inhibitors have been described, but in the majority of cases, it results from reactivation of the MEK/ERK pathway (). Thus, amplification or upregulation of growth factors or receptor tyrosine kinases (RTKs), which signal through the SRC-family kinases (SFKs), can lead to pathway reactivation and resistance. Similarly, acquisition of secondary mutations in NRAS, which signals through CRAF (a close relative of BRAF), can also lead to resistance. In addition, amplification of mutant BRAF or alternative splicing of mutant BRAF mRNA, upregulation of the MEK kinase COT, or mutations in MEK can also drive resistance.

Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K.

Vemurafenib is an orally available and clinically active small-molecule inhibitor of BRAF that achieves increased progression-free and overall survival of patients with BRAF mutant melanoma, but not those with BRAF wild-type melanoma (). However, despite initially impressive responses, most patients treated with vemurafenib develop acquired resistance after a relatively short period of disease control. Furthermore, ∼20% of patients having BRAF mutant melanoma present intrinsic resistance and do not respond to vemurafenib. Thus, resistance is a persistent clinical problem in the management of BRAF mutant melanoma, and second-line treatments are urgently required for patients with both intrinsic and acquired resistance to BRAF inhibitors.

Malignant melanoma is the most deadly form of skin cancer. Current estimations are that each year there are >76,000 cases of melanoma with >9,000 deaths in the U.S. ( www.cancer.org ; American Cancer Society). In 2008, >100,000 cases with 22,000 deaths were estimated in Europe (), and >12,000 cases with ∼1,500 deaths were estimated in Australia ( http://www.melanoma.org.au ; Melanoma Institute Australia). Critically, 43%–50% of melanomas carry somatic mutations in BRAF, and those in another 20% carry mutations in NRAS ( www.sanger.ac.uk/genetics/CGP/cosmic/ ). The mutant proteins are active and constitutively activate the RAS-RAF-MEK-ERK pathway, driving cancer cell proliferation and survival and, thereby, tumor progression.

The aforementioned data show that SFK signaling is increased in the majority of BRAF-inhibitor-resistant tumors, and furthermore, that tumors with increased SFK phosphorylation are sensitive to CCT196969 and CCT241161. However, not all resistant tumors show increased SFK phosphorylation, so we tested CCT196969 and CCT241161 in a PDX from a patient with stage IV BRAF mutant melanoma who achieved a partial response to dabrafenib plus trametinib but relapsed after only 5 months (patient #13; Table S4 ). Again, this patient’s tumors expressed melanoma markers before and after treatment ( Figure S6 A), and critically, although ERK phosphorylation is elevated in this resistant tumor, SFK phosphorylation is not ( Figure 6 A), suggesting that resistance is mediated by events downstream of SFKs. We confirm that the BRAFmutation persists in the resistant tumor, but additionally, we observed an acquired NRASmutation that is not present in the pretreatment tumor ( Figure 6 B). Critically, a PDX from this patient is resistant to dabrafenib plus trametinib but sensitive to CCT196969 and CCT241161 ( Figure 6 C), and no body weight loss was observed in the mice ( Figure S6 B).

(C) Growth of PDX from patient #13 in NSG mice treated with dabrafenib plus trametinib (dab + tram), CCT196969, or CCT241161 21 days after tumor implant. ∗∗∗ p ≤ 0.001 (t test, two-tailed).

(A) pERK and pSFK in PDX from patient #13 before and after treatment with dabrafenib plus trametinib. Scale bars, 100 μm.

Thus, CCT196969 and CCT241161 inhibit both BRAF and SRC. Moreover, they inhibit the growth of PDXs from tumors that are resistant to BRAF inhibitors and have increased pSFK. Critically, we find that SFK phosphorylation is increased, particularly in the plasma membrane, in six of another seven melanomas from patients who presented acquired or intrinsic resistance to vemurafenib ( Figure S5 C; data not shown). Thus, we show that SFK phosphorylation is increased in nine of the ten tumors we examined, confirming the critical role of SRC signaling in resistance.

Subsequently, we tested CCT196969 and CCT241161 in a PDX from a patient with stage IV BRAF mutant melanoma who did not respond to vemurafenib and was diagnosed with progressive disease due to intrinsic resistance (patient # 5; Table S4 ). As before, the tumors from this patient expressed melanoma markers before and after treatment ( Figure S5 A), and ERK and SFK phosphorylation was elevated in the tumors following vemurafenib treatment ( Figure 5 A). Note that cells from this patient’s resistant tumor are more sensitive to CCT196969 and CCT241161 than to PLX4720 ( Figure 5 B), and critically, a PDX from the resistant tumor was more sensitive to CCT196969 and CCT241161 than to PLX4720 ( Figure 5 C). Also in this experiment, we did not observe any loss in body weight in the mice ( Figure S5 B).

(C) Growth of PDX from patient #5 in NSG mice treated with PLX4720, CCT196969, or CCT241161 21 days after tumor implant. ∗∗∗ p ≤ 0.001 (t test, two-tailed).

We also tested CCT196969 and CCT241161 in a PDX from a patient with stage IV BRAF mutant melanoma who had achieved a partial response to vemurafenib but who then relapsed with acquired resistance after only 5 months (patient #4; Table S4 ). Again, we confirm that the tumors from this patient express melanoma markers before and after vemurafenib treatment ( Figure S4 A), that ERK and SFK phosphorylation is elevated in the resistant tumor ( Figure 4 E), and that a PDX from the resistant tumor is resistant to PLX4720 but sensitive to CCT196969 and CCT241161 ( Figure 4 F). Note that also here, CCT196969 and CCT241161 do not cause body weight loss in the mice ( Figure S4 B).

Next, we tested CCT196969 and CCT241161 in a tumor from a patient with stage IV BRAF mutant melanoma who achieved a complete response to vemurafenib but relapsed after 15 months with acquired resistance (patient #3; Table S4 ). We show that tumors from this patient express the melanoma markers HMB45 and S100 before and after treatment ( Figure S4 A). Note that, compared to the pretreatment tumor, ERK and SFK phosphorylation is elevated in the resistant tumor ( Figure 4 A), and cells from the resistant tumor are not inhibited by PLX4720, whereas they are sensitive to CCT196969 and CCT241161 ( Figure 4 B). Furthermore, CCT196969 and CCT241161 inhibit ERK and SRC and induce tumor regression in a PDX from the resistant tumor ( Figures 4 C and 4D), again without causing body weight loss in the mice ( Figure S4 B). Note that PLX4720 does not inhibit ERK or SRC in this PDX ( Figure 4 C), and accordingly, neither does it inhibit the growth of this PDX ( Figure 4 D).

CCT196969 and CCT241161 Inhibit the Growth of PDXs from Patients with Acquired and Intrinsic Resistance to BRAF Inhibitors

Notably, CCT196969 and CCT241161 induce caspase 3 and PARP cleavage, demonstrating that they induce apoptosis, whereas PLX4720 does not ( Figure 3 I). Finally, we show that xenografts grown from the cells of patient #2’s tumor are resistant to PLX4720, whereas CCT196969 and CCT241161 achieve complete inhibition of these xenografts ( Figure 3 J) without causing any body weight loss to the mice ( Figure S3 A).

We also tested whether inhibition of p38 MAPK by CCT196969 and CCT241161 contributed to the inhibition of growth of the cells. We show that the cells are highly resistant to the p38 MAPK inhibitor SB203580 (50% inhibitory concentration [IC] = 29.4 μM) and that SB203580 does not cooperate with the pan-RAF inhibitors RAF265 or MLN2480 to inhibit the growth of the cells ( Figure S3 E). Thus, p38 MAPK does not appear to play a role in regulating the growth of these cells, so inhibition of p38 MAPK by CCT196969 and CCT241161 does not contribute to the inhibition of their growth.

Next, we compared the inhibition of SRC in these resistant cells treated with CCT196969 and CCT241161 or three other pan-RAF inhibitors (RAF265, TAK632, and MLN2480) (K. Galvin, 2012, Am. Assoc. Cancer Res., conference;), or another BRAF inhibitor (ARQ736) (Y.Z. Yu et al., 2010, Am. Assoc. Cancer Res., conference), which have entered clinical trials. Notably, all six compounds inhibit ERK, but only CCT196969 and CCT241161 also inhibit SFKs ( Figure S3 B), and CCT196969 inhibits the growth of the cells more potently than any of the other inhibitors ( Figure S3 C). We also assessed SRC inhibitor 1, a selective SFK inhibitor () in these resistant cells. Although SRC inhibitor 1 is inactive alone, it increases the activity of the pan-RAF inhibitor TAK632 against these cells ( Figure S3 D). This confirms that concurrent inhibition of RAF and SFKs cooperates to inhibit the growth of cells that are resistant to BRAF-selective inhibitors.

We also examined our inhibitors in samples from a second patient (patient #2; Table S4 ), who presented stage IV BRAF mutant metastatic melanoma and achieved a partial response to vemurafenib but relapsed after only 3 months. A cell line derived from a vemurafenib-resistant melanoma is resistant to PLX4720 but sensitive to CCT196969 and CCT241161 ( Figure 3 F), so we treated this cell line and two other cell lines (biological replicates)—derived from two patients who developed resistance to vemurafenib—with PLX4720, CCT196969, or CCT241161 and performed reverse phase protein arrays (RPPAs) to examine the phosphorylation of 25 proteins. For most of the proteins, we did not observe significant differences following treatment with any of the compounds, but MEK, ERK and SRC phosphorylation were strongly suppressed by CCT196969 and CCT241161, but not by PLX4720 ( Figure 3 G; Table S5 ). We confirm that CCT196969 and CCT241161 inhibit MEK, ERK, and SRC in the cells from patient #2, whereas PLX4720 does not ( Figure 3 H).

We tested whether our compounds are active in melanomas that are resistant to BRAF inhibitors. A375 cells that are continually exposed to PLX4720 developed resistance as demonstrated by the regrowth of cells after 20 days, but no cells are able to grow in parallel cultures exposed to CCT196969 or CCT241161 ( Figure 3 A). Note that A375 cells that have developed resistance to PLX4720 following continual exposure to the drug are still sensitive to CCT196969 and CCT241161 ( Figure 3 B), and more important, CCT196969 and CCT241161 inhibit the growth of PLX4720-resistant A375 xenografts (A375/R) in mice ( Figure 3 C), without causing any body weight loss to the mice ( Figure S3 A). Next, we induced resistance to PLX4720 in a patient-derived xenograft (PDX) from a patient (patient #1; Table S4 ) who presented stage III BRAF mutant melanoma and had a tumor removed for palliation. The tumor was propagated in immunocompromised mice, and the mice were then treated with PLX4720 until the tumor developed resistance ( Figures 3 D and S3 A). Note that despite its resistance to PLX4720, this tumor remains sensitive to CCT196969 and CCT241161 ( Figure 3 E).

(E) Growth of PLX4720-resistant PDX from patient #1, from (D), in mice treated with PLX4720, CCT196969, or CCT241161 7 days after cell injection. ∗∗∗ p ≤ 0.001 (t test, two-tailed).

Taken together, the aforementioned data confirm that CCT196969 and CCT241161 are orally available, well-tolerated BRAF inhibitors that directly inhibit BRAFin cells. We show that CCT196969 and CCT241161 are active against melanoma and colorectal cancer cell lines that are mutant for BRAF ( Figures 2 A and 2B ). In addition, unlike the BRAF-selective inhibitors PLX4720 and SB590885, but in common with the MEK inhibitor PD184352, CCT196969 and CCT241161 are also active against RAS mutant melanoma and colorectal cancer cells ( Figures 2 A and 2B). In general, CCT196969 and CCT241161 are not active against cancer cells that are wild-type for BRAF and NRAS, but curiously, SK-Mel 23 cells are sensitive to these compounds ( Figure 2 A). The reasons for this are unclear, but ERK activity is elevated in these cells and sensitive to CCT196969 and CCT241161 ( Figure S2 ), suggesting that their growth depends on this pathway, presumably due to events upstream of RAS. Notably, in contrast to previously described BRAF inhibitors (), CCT196969 and CCT241161 inhibit rather than activate MEK in NRAS mutant cells ( Figure 2 C), and they inhibit NRAS mutant cell growth more efficiently than does PLX4720 ( Figure 2 D). Furthermore, in contrast to the BRAF inhibitor PLX4720, CCT196969 and CCT241161 inhibit the growth of NRAS mutant DO4 tumor xenografts in nude mice ( Figure 2 E). Thus, CCT196969 and CCT241161 are paradox-breaking pan-RAF inhibitors that are active against both BRAF mutant and NRAS mutant melanomas.

(B) Heat map showing sensitivity of cancer cell lines bearing mutations in BRAF, NRAS, or KRAS (shown in the grid below the heat map) presented as GIvalues determined after a 5-day exposure to each compound (BRAF inhibitors PLX4720 and SB590885, MEK inhibitor PD184352, and our compounds CCT241161 and CCT196969) and analysis by sulphorhodamine B staining. Values were log-transformed, and hierarchical clustering was performed with “one minus Pearson correlation” using Gene E ( www.broadinstitute.org/cancer/software/GENE-E/ ).

(A) Cell growth inhibition by CCT196969 or CCT241161 (expressed as log 2 GI 50 in micromolar) in cells carrying BRAF (red), RAS (blue), or neither (green) mutation. ∗ , melanoma cell line; ˆ, colorectal carcinoma cell line; >, breast cancer cell line. WT, wild-type.

To directly test if CCT196969 and CCT241161 are BRAF inhibitors, we replaced the “gatekeeper” threonine 529 (T529) in BRAF with asparagine to block drug binding without compromising kinase activity (). We saw that CCT196969 is 753-fold and CCT241161 is 42-fold less active against BRAFthan BRAF Figure 1 F), demonstrating that the T529N substitution impairs binding of these drugs to BRAF. To test the effects of these mutations in cells, we used Ba/F3 cells. As we have shown previously, Ba/F3 cells grow in an interleukin-3 (IL-3)-dependent manner, but when transformed with BRAFand BRAF, their growth becomes IL-3 independent but dependent on oncogenic BRAF (). Critically, we show that the growth of Ba/F3 cells transformed with BRAFis 48-fold and 19-fold less sensitive to CCT196969 and CCT241161, respectively, than cells transformed with BRAF Figure 1 G), demonstrating directly that these drugs inhibit BRAFin cells.

Oral dosing at 10 mg/kg/day results in plasma concentrations ∼1 μM at 24 hr and 14 hr, for CCT196969 and CCT241161, respectively ( Figure S1 A; Table S1 ), with areas under the curve of ∼416,000 and ∼275,000 nM.hr, respectively. These compounds are equally orally bioavailable at ∼55%, and even at 10 mg/kg/day, we achieve plasma levels well above the half-maximal inhibition of cell proliferation (GI) values for BRAF-selective-inhibitor-resistant cells (GI= 0.4 μM, mean value from Figure 2 A) and NRAS mutant melanoma cells (GI= 0.6 μM, mean value from Figure 2 A). We confirm that doses of 30 mg/kg for 4 days do not cause significant weight loss ( Figure S1 B), so we selected 20 mg/kg/day (7 days/week; no weekend break) based on efficacy and tolerability. Critically, at these doses, we achieve tumor regression with BRAF mutant A375 tumor xenografts in nude mice ( Figure 1 E), although CCT196969 is also effective at 10 mg/kg/day (data not shown). CCT196969 and CCT241161 achieve plasma exposures of ∼40 μM and ∼50 μM, respectively ( Table S2 ), which are similar to those seen for vemurafenib in humans (). Note, also, that 1 hr after the last dose was administered at the end of the therapy experiments, the concentration of drug in the tumors was ∼7 μM for CCT196969 and 6.5–10 μM for CCT241161 ( Table S3 ), levels that are well above the GIvalues for growth inhibition of cancer cells.

A comprehensive safety profile analysis on CCT196969 shows that the compound is extremely well tolerated at the doses assessed and does not produce any significant adverse effects in vivo. A single dose at 20 mg/kg does not produce any clinical signs and produces no observed adverse effects in CD-1 mice. When administered at 40 mg/kg, we observed slight, transient tachypnoea 1 hr after dosing, but no effect on body weight, so 40 mg/kg is defined as the maximum tolerated dose (single dose). At 20 mg/kg daily × 24 days, we observed no clinical signs or body weight loss, and at 25 mg/kg daily for 19 days, we did not observe any mortality, although tachypnoea with decreased activity and excitation were seen. However, as mentioned earlier, the treated group did not show any body weight loss or reduction in food intake. In addition, at the end of the study, microscopical examination of tissues did not identify any treatment-related changes.

As previously described, we have pursued a drug discovery program in which we designed, synthesized, and characterized inhibitors of the inactive conformation of BRAF). Here, we describe two further inhibitors, CCT196969 and CCT241161 ( Figure 1 A; synthesis and characterization are described in the Supplemental Experimental Procedures available online). These compounds were found to inhibit BRAF, CRAF, and SFKs ( Table 1 ). Since resistance to BRAF and BRAF/MEK inhibitors can be driven by RTKs signaling through SFKs, or mutant NRAS signaling through CRAF, we selected these compounds for further study. CCT196969 inhibits BRAF at 100 nM and BRAFat 40 nM, while CCT241161 inhibits BRAF at 252 nM and BRAFat 15 nM ( Table 1 ). CCT196969 inhibits CRAF at 12 nM, SRC at 26 nM, and LCK at 14 nM, while CCT241161 inhibits CRAF at 6 nM, SRC at 15 nM, and LCK at 3 nM ( Table 1 ). Neither compound inhibits MEK1 or the MEK1 kinase COT ( Table 1 ), and, in a panel of protein kinases, they only inhibit SRC, LCK, and the p38 mitogen-activated protein kinases (MAPKs) ( Figure 1 B). Both inhibit MEK and ERK in WM266.4 cells (BRAF mutant), but not D35 cells (BRAF/RAS wild-type) ( Figure 1 C), and both inhibit growth of BRAF mutant melanoma cells more potently than PLX4720, an analog of the BRAF-selective inhibitor vemurafenib that has superior bioavailability in mice () ( Figure 1 D).

(C) Phospho-MEK (pMEK), phospho-ERK (pERK), and ERK2 in WM266.4 and D35 cells treated for 24 hr with DMSO (D), CCT196969, or CCT241161.

Discussion

Here, we describe CCT196969 and CCT241161, BRAF/CRAF inhibitors that are also active against SFKs. These agents block BRAF mutant and NRAS mutant melanoma cell growth in vitro and in vivo. They are active against treatment-naive BRAF mutant tumors, against melanomas that are resistant to BRAF-selective drugs, and against a sample from a patient who was resistant to a BRAF/MEK inhibitor combination. The inhibitors are active in tumors from patients with acquired or intrinsic resistance. Critically, pERK was increased in all of the resistant patient tumors, consistent with resistance being mediated by MEK/ERK pathway activation. SFK phosphorylation was also increased in nine of 11 resistant tumors, but in the patient whose resistance was associated with an acquired mutation in NRAS, SFK phosphorylation was not increased.

Thus, we have developed pan-RAF/SFK inhibitors that are orally available and well tolerated at therapeutic doses. They are active against treatment-naive BRAF and NRAS mutant tumors and against a range of tumors that are resistant to BRAF and BRAF plus MEK inhibitors, critically achieving regressions in a range of tumors. We also note that they were active in PDXs from patients who subsequently failed ipilimumab treatment. For treatment-naive patients, the presence of a BRAF or NRAS mutation may serve as a predictive biomarker to select patients who could benefit from treatment with these inhibitors. For patients whose tumors are resistant to current BRAF and MEK inhibitors, upregulated RTK signaling evidenced by increased SFK phosphorylation or pathway reactivation evidenced by increased ERK phosphorylation may provide predictive biomarkers to select patients for treatment. The presence of an NRAS mutation may also serve as a predictive biomarker for patient selection in the resistant setting. Thus, we posit that these inhibitors could provide first-line therapy for treatment-naive patients and second-line therapy for a range of patients with relapsed melanoma. We aim to conduct phase I clinical trials with this series of inhibitors starting in 2015.