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The effects of selective phosphoinositide 3-kinase (PI3K) and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of receptor tyrosine kinases, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild-type RAS and of RAF–MEK–ERK signaling. Inhibition of RAS–ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death, and in murine models of HER2+ cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regression. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity.Significance: We show that the RAS–ERK pathway is a key downstream effector pathway of oncogenic PI3K. Coordinate downregulation of AKT and ERK is necessary for induction of apoptosis and antitumor activity and can be accomplished with pulsatile dosing, which will likely decrease toxicity and allow administration of therapeutic doses. Cancer Discov; 4(3); 334–47. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 259

Supplementary Figure S1: Representative H&E, phospho-ERK and FISH images of pre-dabrafenib (3) and post-dabrafenib (4) tumors. Supplementary Figure S2: WES analysis of copy number variation in pre- and postdabrafenib tumors. Supplementary Figure S3: Whole copy number profiles from WES of pre- and posttreatment tumors. Supplementary Figure S4: Homology alignment of BRAF p. L514 with residues in other tyrosine kinases. Supplementary Figure S5: Relative frequency of BRAF variant alleles in SK-BT-DR cells determined by individual clone sequencing. Supplementary Figure S6: BRAF V600E/L514V reduces dabrafenib sensitivity in NIH- 3T3 cells, related to Figure 2. Supplementary Figure S7: BRAF V600E/L514V confers biochemical resistance to dabrafenib over a time course, related to Figure 2G. Supplementary Figure S8: Comparison of IC50, IC75 and IC90 of dabrafenib against A375 cells expressing BRAF V600E and BRAF V600E/L514V, related to Figure 2H. Supplementary Figure S9: The BRAF V600E/L514V double mutant promotes homodimerization, related to Figure 3A and B. Supplementary Figure S10: BRAF L514V alone is hypoactive and associated with decreased ERK signaling that is not sensitive to dabrafenib. Supplementary Figure S11: BRAF V600E/L514V is inhibited by dabrafenib in a purified kinase assay, indicating that it is not a gatekeeper mutation. Supplementary Figure S12: Quantitation of p-MEK and p-ERK immunoblots, related to Figure 4B. Supplementary Figure S13: Comparison of IC50, IC75 and IC90 of trametinib against A375 expressing BRAF V600E and BRAF V600E/L514V, related to Figure 4C. Supplementary Figure S14: The BRAF V600E/L514V mutant mediates resistance to dabrafenib that cannot be completely overcome by trametinib or dabrafenib plus trametinib, related to Figure 4D. Supplementary Figure S15: V5, p-MEK, total MEK immunoblots, and quantitation of p-ERK immunoblots, related to Figure 5A. Supplementary Figure S16: Novel RAF dimer inhibitors, MEK inhibitor and ERK inhibitor equipotently inhibit cell growth in BRAF V600E and V600E/L514V expressing cells. Supplementary Figure S17: Novel RAF dimer inhibitor, MEK inhibitor and ERK inhibitor equipotently inhibit ERK signaling in BRAF V600E and V600E/L514V expressing cells. Supplementary Figure S18: BGB3245 binds mutant BRAF V600E monomer and second site of V600E/L514V dimer with similar affinity. Supplementary Table S1: Mutations identified by WES of pre-dabrafenib and postdabrafenib tumors. Supplementary Table S2: Secondary mutations associated with acquired resistance and occurring in residues homologous with L514 in BRAF. Supplementary Table S3: BRAF L514V allele frequency determined by ddPCR.

PDF file 93K, This file contains detailed description of experimental procedures as well as reagents utilized

PDF file 40K, Supplementary Figure S6. Induction of apoptosis by PI3K inhibition in HER2-amplified cells. Related to main Figure 6

PDF file 67K, This file contains legends for supplementary figures S1-S7. It describes in detail what's included in each figure

Table S1: AML Patient Characteristics Table S2: Plasma sample characteristics

Figure S1: Sorafenib treatment induces phospho-ERK rebound in 32D/ITD cells. Figure S2: Further characterization of FLT3 inhibitor-mediated signaling adaptation. Figure S3: FLT3 signaling is maintained in low serum conditions. Figure S4: MEK inhibitor treatment abrogates phospho-ERK rebound. Figure S5: MEK inhibition synergizes with FLT3 inhibition to increase apoptosis in FLT3/ITD AML. Figure S6: NRAS mutant AML is sensitive to MEK inhibition but insensitive to the addition of sorafenib. Figure S7: MEK inhibition sensitizes K562 but does not sensitize PC9 or SKBR3 cells to RTK inhibition.

FMS-like tyrosine kinase-3 (FLT3) tyrosine kinase inhibitors (TKI) have been tested extensively to limited benefit in acute myeloid leukemia (AML). We hypothesized that FLT3/internal tandem duplication (ITD) leukemia cells exhibit mechanisms of intrinsic signaling adaptation to TKI treatment that are associated with an incomplete response. Here, we identified reactivation of ERK signaling within hours following treatment of FLT3/ITD AML cells with selective inhibitors of FLT3. When these cells were treated with inhibitors of both FLT3 and MEK in combination, ERK reactivation was abrogated and anti-leukemia effects were more pronounced compared with either drug alone. ERK reactivation was also observed following inhibition of other tyrosine kinase–driven cancer cells, including EGFR-mutant lung cancer, HER2-amplified breast cancer, and BCR–ABL leukemia. These studies reveal an adaptive feedback mechanism in tyrosine kinase–driven cancers associated with reactivation of ERK signaling in response to targeted inhibition. Cancer Res; 77(20); 5554–63. ©2017 AACR.

BRAFV600E hyperactivates ERK and signals as a RAF inhibitor–sensitive monomer. Although RAF inhibitors can produce impressive clinical responses in patients with mutant BRAF tumors, the mechanisms of resistance to these drugs are incompletely characterized. Here, we report a complete response followed by clinical progression in a patient with a BRAFV600E-mutant brain tumor treated with dabrafenib. Whole-exome sequencing revealed a secondary BRAFL514V mutation at progression that was not present in the pretreatment tumor. Expressing BRAFV600E/L514V induces ERK signaling, promotes RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Newer RAF dimer inhibitors and an ERK inhibitor are effective against BRAFL514V-mediated resistance. Collectively, our results validate a novel biochemical mechanism of RAF inhibitor resistance mediated by a secondary mutation, emphasizing that, like driver mutations in cancer, the spectrum of mutations that drive resistance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specific prevalence. Significance: In contrast to receptor tyrosine kinases, in which secondary mutations are often responsible for acquired resistance, second-site mutations in BRAF have not been validated in clinically acquired resistance to RAF inhibitors. We demonstrate a secondary mutation in BRAF (V600E/L514V) following progression on dabrafenib and confirm functionally that this mutation is responsible for resistance. Cancer Discov; 8(9); 1130–41. ©2018 AACR. See related commentary by Romano and Kwong, p. 1064. This article is highlighted in the In This Issue feature, p. 1047

BRAF

Mammalian species differ dramatically in telomere biology. Species larger than 5–10 kg repress somatic telomerase activity and have shorter telomeres, leading to replicative senescence. It has been proposed that evolution of replicative senescence in large-bodied species is an anti-tumour mechanism counteracting increased risk of cancer due to increased cell numbers. By contrast, small-bodied species express high telomerase activity and have longer telomeres. To counteract cancer risk due to longer lifespan, long-lived small-bodied species evolved additional telomere-independent tumour suppressor mechanisms. Here, we tested the connection between telomere biology and tumorigenesis by analysing the propensity of fibroblasts from 18 rodent species to form tumours. We found a negative correlation between species lifespan and anchorage-independent growth. Small-bodied species required inactivation of Rb and/or p53 and expression of oncogenic H-Ras to form tumours. Large-bodied species displayed a continuum of phenotypes requiring additional genetic ‘hits’ for malignant transformation. Based on these data we refine the model of the evolution of tumour suppressor mechanisms and telomeres. We propose that two different strategies evolved in small and large species because small-bodied species cannot tolerate small tumours that form prior to activation of the telomere barrier, and must instead use telomere-independent strategies that act earlier, at the hyperplasia stage. This article is part of the theme issue ‘Understanding diversity in telomere dynamics’.

FMS-like tyrosine kinase-3 (FLT3) tyrosine kinase inhibitors (TKI) have been tested extensively to limited benefit in acute myeloid leukemia (AML). We hypothesized that FLT3/internal tandem duplication (ITD) leukemia cells exhibit mechanisms of intrinsic signaling adaptation to TKI treatment that are associated with an incomplete response. Here, we identified reactivation of ERK signaling within hours following treatment of FLT3/ITD AML cells with selective inhibitors of FLT3. When these cells were treated with inhibitors of both FLT3 and MEK in combination, ERK reactivation was abrogated and anti-leukemia effects were more pronounced compared with either drug alone. ERK reactivation was also observed following inhibition of other tyrosine kinase–driven cancer cells, including EGFR-mutant lung cancer, HER2-amplified breast cancer, and BCR–ABL leukemia. These studies reveal an adaptive feedback mechanism in tyrosine kinase–driven cancers associated with reactivation of ERK signaling in response to targeted inhibition. Cancer Res; 77(20); 5554–63. ©2017 AACR.

BRAF V600E hyperactivates ERK and signals as an RAF inhibitor-sensitive monomer. While RAF inhibitors can produce impressive clinical responses in patients with mutant BRAF tumors, the mechanisms of resistance to these drugs are incompletely characterized. Here, we report a complete response followed by clinical progression in a patient with a BRAF V600E-mutant brain tumor treated with dabrafenib. Whole-exome sequencing revealed a secondary BRAF L514V mutation at progression that was not present in the pretreatment tumor. Expressing BRAF V600E L514V induces ERK signaling, promotes RAF dimer formation, and is sufficient to confer resistance to dabrafenib. A novel class of RAF dimer inhibitors and an ERK inhibitor are effective against BRAF L514V-mediated resistance. Collectively, our results validate a novel biochemical mechanism of RAF inhibitor resistance mediated by a secondary mutation, emphasizing that like driver mutations in cancer, the spectrum of mutations that drive resistance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specific prevalence. Citation Format: Jiawan Wang, Zhan Yao, Philip Jonsson, Amy Allen, Alice Can Ran Qin, Sharmeen Uddin, Ira J. Dunkel, Mary Petriccione, Katia Manova, Sofia Haque, Marc Rosenblum, David J. Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas. A secondary mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3184.

BRAF V600E drives tumors by hyperactivating ERK signaling, is activated in a RAS-independent manner, and signals as a monomer. Selective inhibitors of RAF potently inhibit BRAF V600E and downstream ERK signaling, and have been associated with impressive clinical responses in patients whose tumors harbor V600E mutations in BRAF. Almost all patients, however, ultimately develop resistance. To date, acquired resistance mechanisms validated in patient samples have included splice forms of BRAF, mutations in NRAS, and BRAF amplification, all of which promote formation of RAF dimers, and mutations in MEK. Second-site point mutations in BRAF that promote dimer formation and are associated with acquired resistance have not previously been identified in RAF inhibitor-resistant tumors. Here, we report a complete response followed by clinical progression of a BRAF V600E-mutant pediatric brain tumor treated with the RAF inhibitor dabrafenib. We identified a second-site mutation in BRAF at the time of progression that was not present in the pretreatment tumor. Further study indicated the acquired mutation is in cis with V600E. We demonstrate that the acquired resistance mutation induces RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Moreover, we showed that ERK signaling activated by the double mutation is sensitive to MEK inhibition or to the RAF plus and MEK inhibitor combination. We also find that two novel RAF dimer inhibitors may overcome resistance mediated by the novel mutation in BRAF. This is the first study, to our knowledge, that demonstrates the emergence of a confirmed second-site mutation in BRAF V600E and validates that this mutation is responsible for acquired resistance in an initially responding patient treated with dabrafenib. The mechanism of acquired resistance can be abrogated by the novel RAF dimer inhibitors. Our data confirm that a novel class of RAF dimer inhibitors are active against an acquired resistance mutation, suggesting an improved personalized treatment option for patients who harbor similar second-site mutations in BRAF. Citation Format: Jiawan Wang, Zhan Yao, Philip Jonsson, Amy Allen, Alice Can Ran Qin, David Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas. A second-site mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A129.

The effects of selective phosphoinositide 3-kinase (PI3K) and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of receptor tyrosine kinases, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild-type RAS and of RAF–MEK–ERK signaling. Inhibition of RAS–ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death, and in murine models of HER2+ cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regression. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity. Significance: We show that the RAS–ERK pathway is a key downstream effector pathway of oncogenic PI3K. Coordinate downregulation of AKT and ERK is necessary for induction of apoptosis and antitumor activity and can be accomplished with pulsatile dosing, which will likely decrease toxicity and allow administration of therapeutic doses. Cancer Discov; 4(3); 334–47. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 259

FMS-like tyrosine kinase-3 (FLT3) is one of the most commonly mutated genes in acute myeloid leukemia (AML). The internal tandem duplication (FLT3/ITD) is an established driver mutation in AML and results in constitutive activation of the receptor, activating downstream signaling pathways including STAT5, PI3K/AKT, and RAF/MEK/ERK. The use of FLT3 tyrosine kinase inhibitors (TKIs) for the treatment of FLT3/ITD AML has been explored as a promising strategy for over a decade, but clinical responses have remained limited. In some cancers driven by mutated oncogenes, small molecule inhibition of the target is known to result in reactivation of downstream signaling pathways. This response has been observed in the context of PI3K/AKT/mTOR and BRAF inhibition, but it remains unclear whether this phenomenon extends to cancers driven by receptor tyrosine kinase (RTK) activation, such as FLT3/ITD AML. We hypothesized that FLT3/ITD leukemia cells exhibit mechanisms of intrinsic signaling adaptation to FLT3 TKI treatment that are associated with an incomplete biologic response. To test this, we treated FLT3/ITD AML cell lines (Molm14 and MV4;11) with FLT3 TKIs for up to 48 hours. We observed a rebound in ERK phosphorylation beginning as early as 6 hours and continuing for the duration of treatment, while no such rebound was observed in the phosphorylation of downstream targets AKT or STAT5. When these cells were treated with inhibitors of both FLT3 and MEK in combination, little to no pERK rebound was observed, and the anti-leukemia effects were more pronounced compared to either drug alone, both in vitro and in vivo. In vitro, the addition of low dose MEK inhibitor to FLT3 TKI treatment synergistically increased cell death and decreased cell proliferation. In a xenograft transplant model of leukemia, the addition of low-dose MEK inhibitor to FLT3 TKI treatment resulted in a significant reduction of both peripheral blood and bone marrow blasts (p < 0.05). We next sought to determine whether rebound in ERK signaling following FLT3 inhibition was likely to occur in patients as well. We first examined whether primary leukemic cells from patients with FLT3/ITD AML also exhibit the same phenomenon observed in FLT3/ITD cell lines. After 24 hours of sorafenib treatment, the majority of samples revealed a similar rebound in ERK phosphorylation, despite sustained inhibition of phospho-STAT5. As observed with the cell lines, the addition of a MEK inhibitor to sorafenib treatment resulted in sustained pERK inhibition after 24 hours. Next, we explored whether signaling adaptation observed in culture conditions would be recapitulated by the concentrations of sorafenib present in human plasma from patients being treated with this FLT3 TKI. To replicate these conditions, we cultured Molm14 cells in plasma samples obtained from AML patients treated with sorafenib for a prolonged period. Using a variant of the plasma inhibitory activity assay, Molm14 cells were treated in either normal human plasma for one hour or patient plasma for either one or 24 hours. For all plasma samples, profound pERK rebound was observed by 24 hours after treatment, despite maximal inhibition at one hour and persistent inhibition of FLT3 and STAT5 phosphorylation over the duration. Finally, we sought to determine whether TKI-mediated rebound in ERK activity was restricted to FLT3/ITD AML or may also occur in the context of other RTK-driven cancers, such as those driven by EGFR mutation or HER2 amplification. We treated an EGFR mutant lung cancer (PC-9) and a HER2 amplified breast cancer (SKBR3) cell line with the EGFR/HER2 inhibitor lapatinib. As was seen for FLT3/ITD driven AML, pERK rebound was observed following 24 hours of TKI treatment despite persistent inhibition of the receptor and downstream target AKT. Again, the rebound could be overcome with the addition of low concentrations of MEK inhibitor. Together, these studies reveal that FLT3/ITD and other RTK driven cancer cells demonstrate an adaptive feedback mechanism capable of reactivating ERK signaling in response to upstream target inhibition. This adaptation limits the efficacy of TKI treatment and can be abrogated by the addition of a MEK inhibitor. Our data suggest that the addition of low-dose MEK inhibitor to TKI treatment as a means to overcome signaling adaptation may improve outcomes for patients with FLT3/ITD AML and possibly other RTK-driven cancer types. Disclosures Levis: Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria.