The paradox-breaking panRAF plus SRC family kinase inhibitor, CCT3833, is effective in mutant KRAS-driven cancers

Background KRAS is mutated in ∼90% of pancreatic ductal adenocarcinomas, ∼35% of colorectal cancers and ∼20% of non-small-cell lung cancers. There has been recent progress in targeting G12CKRAS specifically, but therapeutic options for other mutant forms of KRAS are limited, largely because the complexity of downstream signaling and feedback mechanisms mean that targeting individual pathway components is ineffective. Design The protein kinases RAF and SRC are validated therapeutic targets in KRAS-mutant pancreatic ductal adenocarcinomas, colorectal cancers and non-small-cell lung cancers and we show that both must be inhibited to block growth of these cancers. We describe CCT3833, a new drug that inhibits both RAF and SRC, which may be effective in KRAS-mutant cancers. Results We show that CCT3833 inhibits RAF and SRC in KRAS-mutant tumors in vitro and in vivo, and that it inhibits tumor growth at well-tolerated doses in mice. CCT3833 has been evaluated in a phase I clinical trial (NCT02437227) and we report here that it significantly prolongs progression-free survival of a patient with a G12VKRAS spindle cell sarcoma who did not respond to a multikinase inhibitor and therefore had limited treatment options. Conclusions New drug CCT3833 elicits significant preclinical therapeutic efficacy in KRAS-mutant colorectal, lung and pancreatic tumor xenografts, demonstrating a treatment option for several areas of unmet clinical need. Based on these preclinical data and the phase I clinical unconfirmed response in a patient with KRAS-mutant spindle cell sarcoma, CCT3833 requires further evaluation in patients with other KRAS-mutant cancers.


INTRODUCTION
Lung cancer [w90% of which are non-small-cell lung cancers (NSCLC)] is the most common cancer worldwide and in the UK has a 5-year overall survival (OS) of only 15%. 1,2 Colorectal cancer (CRC) is the third most common cancer 1 and in the UK has a 5-year OS of 60% 2 , and pancreatic ductal adenocarcinoma (PDAC) is the tenth most common cancer in the UK and has the poorest prognosis with 5-year OS of only 5%. 2 These poor survival rates are partly due to a lack of treatment options. Surgery is the preferable treatment of NSCLC, PDAC and CRC, but most patients present late with inoperable advanced disease and so receive systemic therapy. 3,4 Targeted therapies are licensed for NSCLC (EGFR, ALK, ROS1 indications) and CRC (KRAS wild-type), but KRAS-mutated cancer remains an area of unmet clinical need. Critically, KRAS is mutated in w20% NSCLC, w90% PDAC and w35% CRC. 5 These patients receive conventional chemotherapy or immunotherapy, often with limited efficacy and potential toxicity 4,6,7 except for KRASmutant NSCLC patients who benefit from immune checkpoint inhibitors compared with KRAS wild-type patients. 8,9 Thus, although RAS (KRAS, NRAS, HRAS) is mutated in w25% of all cancers, treatment of these patients is challenging. 10 Notably, direct inhibitors of KRAS are limited to the p.G12C KRAS-mutant, [11][12][13] so an alternative is to target downstream effectors in the RAF/MEK/ERK pathway, which has led to the development of RAF, MEK and ERK drugs. 14 However, in KRAS-mutant cells, BRAF-selective drugs such as vemurafenib (PLX4032) and dabrafenib 15 cause paradoxical hyperactivation of the RAF-ERK pathway through formation of BRAF-CRAF homo-and hetero-dimers. 16 Unfortunately, targeting MEK downstream of RAF with drugs such as trametinib 17 is ineffective in KRAS-mutant cancers because of feedback mechanisms 18 and adverse side-effects, 19 and therefore these drugs have been unsuccessful in KRAS-mutant PDAC, CRC and NSCLC. 20,21 With a pressing need for different approaches for KRASmutant cancers, here we describe a new drug for this indication. The protein kinase SRC is a master regulator of cancer cell proliferation, metastasis and invasion, and it also potentiates cancer processes such as neo-angiogenesis. 22 SRCfamily kinases (SFKs) are associated with pathogenesis of many cancers, particularly late-stage disease, where its increased activity and expression are associated with disease progression and poorer prognosis. Critically, SRC is a validated target in KRAS-mutant CRC, 23 PDAC 22,24 and NSCLC 22,25 and it is known that G12C KRAS and SRC inhibitors work synergistically to inhibit G12C KRAS NSCLC cell proliferation. 26 In this study, we describe CCT3833, a new combined panRAF and SRC inhibitor. We show that CCT3833 does not drive paradoxical activation of the RAF/MEK/ERK pathway in KRAS-mutant cells and that its ability to exert dual inhibition of RAF and SRC provides effective therapy in preclinical KRAS-mutant PDAC, CRC and NSCLC models. We show that CCT3833 is superior to single-agent panRAF or SRC inhibitors and comparable with combination panRAF þ SRC inhibitors in standard two-dimensional tissue culture and more significantly, in three-dimensional spheroids, which are of intermediate complexity between standard monolayer cultures in vitro and tumors in vivo.
Importantly, CCT3833 has been investigated in a phase I dose-escalation clinical trial (NCT02437227) including 31 patients with solid tumors, of whom at least 10 were known to be KRAS-mutant. We report an unconfirmed partial response and prolonged clinical benefit from CCT3833 in one of these patients, diagnosed with a KRAS-mutant spindle cell sarcoma. This was the only patient with an unconfirmed partial response on trial. Spindle cell sarcomas are connective tissue tumors characterized by spindleshaped cells, and are typically treated with anthracyclines, but with limited and variable responses. 27 Here, we describe a patient with a spindle cell sarcoma presenting a p.G12V KRAS mutation. The patient displayed early disease progression following surgical resection, was not a candidate for doxorubicin chemotherapy and did not respond to the multikinase inhibitor pazopanib. Despite being in the dose escalation phase, CCT3833 achieved a progression-free survival (PFS) of >10 months, and we provide comprehensive analysis of the mechanism of action of CCT3833 in KRAS-mutant cancers to reveal how this patient and others could benefit from this agent.

Cell culture
Cell lines were cultured under standard conditions. Human PDAC cell lines (except for MIA-PaCa2) were a gift from Dr Claus Jorgensen, Calu-1 and H460 cells were a gift from Dr Michela Garofalo and H2009 NSCLC cells were a gift from Dr John Brognard. All other human cell lines were from the American Type Culture Collection (ATCC). Short tandem repeat profiles were routinely compared with known ATCC fingerprints and cells were routinely ensured to be mycoplasma free by PCR. Mouse KPC PDAC cells were a gift of Professor Owen Sansom, or were isolated and established in house from transgenic mice as described. 24 Cells were cultured in Dulbecco's modified Eagle's Medium or RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Short-term growth inhibition assays, long-term cell proliferation assays and tumor spheroid assays were carried out as detailed in Supplementary

Statistics
For graphs, mean values are shown and error bars represent standard error of the mean unless stated otherwise, (*) P 0.05 (Student's or Welch's t-test as indicated). All in vitro experiments were carried out in triplicate unless otherwise stated. Docking studies predict that CCT3833 is a type II inhibitor that binds to the inactive 'DFG-out' conformation of BRAF, 28 and to CRAF and SRC through similar mechanisms ( Figure 1D-F). Specifically, the pyridopyrazinone moiety is predicted to interact with the kinase hinge, the central aromatic ring occupies the ATP binding pocket and the pyrazole ring elaborates into an allosteric site created by the DFG moving into the 'out' conformation ( Figure 1D-F). Moreover, the tert-butyl group is predicted to elaborate into a hydrophobic pocket and the terminal fluorosubstituted phenyl ring points towards the activation loop in all three kinases ( Figure 1D-F). These binding similarities indicate how CCT3833 inhibits both RAF and SRC and we confirm that CCT3833 inhibits both ERK (ppERK; downstream of CRAF) and SFK (ppSFK) phosphorylation in a dosedependent manner in HCT-116 (CRC), A549 (NSCLC) ( Figure 1G

CCT3833 inhibits KRAS-mutant cancer cell growth
The data above show that CCT3833 is a panRAF inhibitor that also inhibits SRC. Notably, RAF and SRC are validated targets in RAS-mutant cancers, because RAF signals downstream of oncogenic KRAS, and SFKs drive cancer cell proliferation and survival. Accordingly, we show that CCT3833 is active against a panel of KRAS-mutant PDAC, CRC and NSCLC cell lines, whereas it is less potent against KRAS/BRAF wild-type cells ( Figure 2A, Supplementary Table S1, available at https://doi. org/10.1016/j.annonc.2020.10.483). Moreover, compared with other RAF inhibitors, in short-term growth assays CCT3833 inhibits HCT-116 growth more potently than the clinically evaluated panRAF inhibitors TAK-632, ARQ736 and MLN-2480 ( Figure 2B, Supplementary Figure S3A, available at https://doi.org/10.1016/j.annonc.2020.10.483). We also show that CCT3833 is more effective than the multikinase inhibitor sorafenib or the BRAF-mutant selective inhibitors PLX4720 and dabrafenib, and that only the MEK inhibitor Although the panRAF inhibitor TAK-632, and the BRAF inhibitors PLX4720 and dabrafenib inhibit BRAF more potently than CCT3833 in in vitro enzyme assays, CCT3833 is more potent at inhibiting KRAS-mutant cancer cell growth, so we examine downstream signaling. In HCT-116, SW620, A549, MIA-PaCa2 and Calu-1 cells, PLX4720 induces paradoxical activation of the ERK pathway and although sorafenib and TAK-632 inhibit ppERK, they are less potent than CCT3833 in their ability to do so ( Figure 2E

RAF and SFK must both be inhibited to block KRAS-mutant cancer growth
Thus, CCT3833 inhibits both CRAF and SRC in KRAS-mutant cancers and so we investigate the contribution of these two activities to the inhibition of long-term cell growth. We show that CCT3833 induces significant caspase-3/7 activation, whereas PLX4720, sorafenib, trametinib and TAK-632 do not activate caspase-3/7 to the same extent ( Figure 3A

Annals of Oncology
(Supplementary Figure S4, available at https://doi.org/10. 1016/j.annonc.2020.10.483). Note also that the inhibitors are used at doses to reflect safe plasma exposure achievable in vivo. Thus, trametinib is used at 20-30 nM, the maximum tolerated patient plasma concentration, 17,29 whereas CCT3833 is used at 1 mM, below the welltolerated mouse plasma concentration in vivo (vide infra).
To assess whether it is necessary to inhibit both ERK and SRC pathways in KRAS-mutant cells, we combine known panRAF and SRC inhibitors. As shown earlier, TAK-632 inhibits ppERK but not ppSFK and conversely, we show that the SRC inhibitor saracatinib inhibits ppSFK but not ppERK ( Figure 4A). Moreover, together these agents mimic the effects of CCT3833 ( Figure 2E) and inhibit both ppERK and ppSFK ( Figure 4A). In long-term clonogenic growth assays, neither TAK-632 nor saracatinib alone inhibit colony formation, whereas together they do inhibit colony formation, both in HCT-116 cells and in H23 lung adenocarcinoma cells, again mimicking the effects of CCT3833 alone ( Figure 4B, Supplementary Figure S5A, available at https://doi.org/10. 1016/j.annonc.2020.10.483). We also assess another SRC inhibitor, bosutinib. Alone, bosutinib does not activate caspase-3/7, but it co-operates with TAK-632 to activate these pro-apoptosis enzymes ( Figure 4C) mimicking the effect of CCT3833 alone. Moreover, TAK-632 and bosutinib co-operate to inhibit the short-term growth of HCT-116, SW620 ( Figure 4D-E), A549 and MIA-PaCa2 cells (Supplementary Figure S5B, available at https://doi.org/ 10.1016/j.annonc.2020.10.483), again mimicking the effect of CCT3833 alone. Notably, the combinations of saracatinib plus TAK-632 or bosutinib plus TAK-632 both inhibit the growth of SW620 tumor spheroids similarly to CCT3833 alone, whereas the response to the single agents is significantly less ( Figure 4F).

CCT3833 inhibits KRAS-mutant tumor growth
Next, we assess CCT3833 in vivo. We show that CCT3833 has good oral bioavailability in mice, excellent pharmacokinetic properties ( Figure 5A, Supplementary  Figure 2A).
We tested CCT3833 in a mouse model of PDAC driven by oncogenic KRAS and inactivating mutation of the tumor suppressor TP53 (KPC cells 24 ). We confirm, commensurate with our human cell observations, that, CCT3833 is more effective than the other pathway inhibitors apart from trametinib at blocking KPC cell growth in short-term proliferation assays, but only CCT3833 completely abrogates growth of these cells in long-term assays ( Figure 5B and C, Supplementary Figure S6A

CCT3833 improves progression-free survival in a patient with G12V KRAS spindle cell sarcoma
A previously fit patient aged in their 70s presented with a 1 year history of non-specific symptoms and was diagnosed with a large intra-abdominal mass associated with the pancreas and invading into the liver parenchyma ( Figure 6A). This was resected and histopathological assessment revealed a lobulated tumor composed of illdefined fascicles of spindle cells, with oval to elongated  moderately pleomorphic nuclei, pale eosinophilic cytoplasm and up to 9 mitoses per 10 high powered fields, later classified as a spindle cell sarcoma NOS (not otherwise specified). Immunohistochemistry was diffusely positive for CD34, but negative for other markers including S-100, SOX10, DOG1, CD117, SMA, desmin, AE1-3, EMA, CD21 and CD23. Moreover, the tumor was negative for the NAB2-STAT6 fusion transcripts.
One year after tumor resection, the patient presented with a multifocal intra-abdominal recurrence, which was inoperable. Although it was later diagnosed as spindle cell sarcoma, due to some histopathological features, it was treated as a malignant solitary fibrous tumor. The patient commenced on the multikinase inhibitor pazopanib, but had extensive disease progression at first radiological assessment after only 12 weeks of treatment ( Figure 6A and B, Table 1 (Table 1). Hypodensity within the lesion seen on scan at cycle 6 and 8 suggests necrosis due to treatment response (white arrowhead). NOS, not otherwise specified; PD, progressive disease; unconfirmed PR, unconfirmed partial response; SD, stable disease.

Annals of Oncology
a day, continuous dosing). Scans were taken at baseline and every 8 weeks during CCT3833 treatment ( Figure 6B). In stark contrast to the progression seen with pazopanib, each of the scans after commencing CCT3833 treatment show stable disease with achievement of an unconfirmed partial response after eight cycles as defined by RECIST 1.1 ( Figure 6B, Table 1, Supplementary Table S4, available at https://doi.org/10.1016/j.annonc.2020.10.483). As a non-RECIST progression was seen on imaging after cycle 10, the patient underwent intrapatient dose escalation to 300 mg once daily, but a scan on cycle 11 (Table 1) confirmed RECIST disease progression and the patient discontinued treatment on day 17 of cycle 12.

DISCUSSION
Herein, we report that the new inhibitor, CCT3833, mediated an unconfirmed partial response in a patient with aggressive KRAS-mutant spindle cell sarcoma who was not eligible for surgery or chemotherapy, and who did not respond to the multikinase inhibitor pazopanib. Despite being in the dose-escalation phase of the clinical trial (NCT02437227), at 75 mg p.o. qd continuous dosing of CCT3833, the patient achieved progression-free survival for 8 months, and did not progress until the 12th cycle of treatment. Together with our preclinical data, this indicates that CCT3833 has potential for the treatment of KRASmutant tumors. Specifically, our preclinical data demonstrate that CCT3833 is effective in KRAS-mutant CRC, NSCLC and PDAC due to its dual anti-panRAF plus anti-SRC activity. RAF is a validated target directly downstream of oncogenic RAS, and SRC is also a validated therapeutic target in CRC, NSCLC and PDAC, where it is hyperactivated and drives cell proliferation and metastasis. [22][23][24][25] Accordingly, SRC inhibitors cooperate with drugs that target the EGFR/RAS pathway. 26,30,31 Our findings that CCT3833 inhibits the growth of KRASdriven murine PDAC in vitro, and in vivo, are aligned to the literature, validating SRC as a therapeutic target in PDAC, where its overexpression or hyperactivation are markers of poor clinical outcome. 24 Moreover, SRC inhibitors are active in preclinical PDAC models and achieve minor clinical responses in PDAC patients. 22 SRC is similarly overexpressed or hyperactivated in CRC 23 and moreover, BRAF-mutant CRC cells can switch between RAF/MEK/ERK and receptor tyrosine kinase signaling, 32 so cell growth is only prevented when both pathways are inhibited. This plasticity may explain the shorter duration of response to BRAF and MEK inhibitors in CRC 21 and may also underpin why mutant KRAS opposes the antitumor effects of EGFR inhibitors in CRC. 33 Notably, MEK and EGFR inhibitors cooperate to block EGFR inhibitor-resistant CRC tumor growth, 33 and we propose therefore that CCT3833 is effective in CRC because it targets the two key pathways downstream from mutant RAS and the hyperactivated receptor tyrosine kinases such as EGFR. Similarly, synergistic efficacy has been shown in vivo by inhibiting the MAPK pathway plus SRC in KRAS/ PIK3CA double-mutant CRC cells. 34 Finally, SRC and ERK signaling are both critical for the growth of KRAS-mutant NSCLC. 20,25 Clinical trials with trametinib in KRAS-mutant NSCLC patients, alone or in combination with chemotherapy, show that single agent MEK inhibitors achieve no improvement compared with chemotherapy and that toxicity limits their clinical use in combination. 19,20 Again, we posit that CCT3833 is effective in NSCLC because of its ability to simultaneously inhibit SRC and ERK signaling. Critically, CCT3833 mediates tumor regression in G12S KRAS-mutant NSCLC xenografts, so it could be considered for treatment of KRAS-mutant NSCLC patients who fail chemotherapy and/or immunotherapy.
In summary, we describe the discovery of CCT3833, a new panRAF/SRC inhibitor, and show that it is effective in KRAS-mutant cancer models, because RAF and SRC are central nodes in KRAS-mutant cancers. CCT3833 differs from the RAF dimer inhibitor LY3009120 35 because it also inhibits SRC and is effective in PDAC. We posit that CCT3833 inhibits tumor growth in RAS-mutant models through on-target inhibition of BRAF and CRAF, and additional on-target inhibition of SRC. Critically, CCT3833 induces tumor cell death and elicits therapeutic efficacy at well-tolerated doses in mice, and it is evaluated in patients in a phase I clinical trial, achieving a proof-of-concept unconfirmed clinical response in a patient with aggressive KRAS-mutant spindle cell sarcoma who was not eligible for other treatments. Taken together, our data support the further clinical evaluation of CCT3833 in patients with KRAS-mutant cancers.