Yogindra Persaud

RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E)

Activated RAS promotes dimerization of members of the RAF kinase family. ATP-competitive RAF inhibitors activate ERK signalling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E). However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4–8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS–GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner

Tumors with mutant BRAF and some with mutant RAS are dependent upon ERK signaling for proliferation, and their growth is suppressed by MAPK/ERK kinase (MEK) inhibitors. In contrast, tumor cells with human EGF receptor (HER) kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors, the RAF inhibitor PLX4032 inhibits the proliferation of BRAFV600E tumor cells but not that of HER kinase-dependent tumors. However, tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to PLX4032. MEK inhibitors inhibit ERK phosphorylation in all normal and tumor cells, whereas PLX4032 inhibits ERK signaling only in tumor cells expressing BRAFV600E. In contrast, the drug activates MEK and ERK phosphorylation in cells with wild-type BRAF. In BRAFV600E tumor cells, MEK and RAF inhibitors affect the expression of a common set of genes. PLX4032 inhibits ERK signaling output in mutant BRAF cells, whereas it transiently activates the expression of these genes in tumor cells with wild-type RAF. Thus, PLX4032 inhibits ERK signaling output in a mutant BRAF-selective manner. These data explain why the drug selectively inhibits the growth of mutant BRAF tumors and suggest that it will not cause toxicity resulting from the inhibition of ERK signaling in normal cells. This selectivity may lead to a broader therapeutic index and help explain the greater antitumor activity observed with this drug than with MEK inhibitors.

Genomic and Biological Characterization of Exon 4 KRAS Mutations in Human Cancer

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.

Concurrent loss of the PTEN and RB1 tumor suppressors attenuates RAF dependence in melanomas harboring V600E BRAF

Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring V600EBRAF mutations. RB1 alterations were mutually exclusive with loss of p16INK4A, suggesting that whereas p16INK4A and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.

NF2 Loss Promotes Oncogenic RAS-Induced Thyroid Cancers via YAP-Dependent Transactivation of RAS Proteins and Sensitizes Them to MEK Inhibition

UNLABELLED Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation is insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacologic disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability. SIGNIFICANCE Intensification of mutant RAS signaling through copy-number imbalances is commonly associated with transformation. We show that NF2/merlin inactivation augments mutant RAS signaling by promoting YAP/TEAD-driven transcription of oncogenic and wild-type RAS, resulting in greater MAPK output and increased sensitivity to MEK inhibitors.

Abstract LB-428: RAS-independent dimerization of BRAF(V600E) splicing variants promotes resistance to RAF inhibitors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Over 50% of melanomas harbor activating mutations in BRAF, most commonly BRAF(V600E). Profound clinical activity has been observed with RAF inhibitors, including vemurafenib, which is now FDA approved for the treatment of patients with advanced melanomas whose tumors harbor a BRAF(V600E) mutation. RAF inhibitors affect ERK signaling in a mutation-specific manner: they inhibit ERK signaling in cells with BRAF(V600E), but paradoxically activate ERK signaling in cells with wild-type BRAF. We recently elucidated the mechanism of this phenomenon: activation of RAS promotes the dimerization of members of the RAF family. At non-saturating concentrations, binding of an ATP-competitive RAF inhibitor to one member of the dimer inhibits it, while also causing its transition to the activated state. This is associated with the allosteric transactivation of the other, non-drug bound member of the dimer. This leads to an overall increase in RAF specific activity and induction of ERK signaling. In BRAF(V600E) melanomas, RAS-GTP levels are low, BRAF(V600E) is found primarily as a monomer and RAF inhibitors effectively inhibit active BRAF(V600E) monomers. This model of RAF transactivation by RAF inhibitors predicts that any molecular lesion that enhances RAF dimerization will promote resistance to RAF inhibitors. To investigate mechanisms of acquired resistance to RAF inhibitors, we treated a sensitive BRAF(V600E) expressing melanoma cell line (SKME239) with the RAF inhibitor vemurafenib for 8 weeks and selected for resistant clones. We found that a subset of cells resistant to vemurafenib expressed a splicing variant form of BRAF(V600E) that lacked exons 4-8, a region that encompasses the RAS-binding domain. This form of BRAF(V600E) had a size of approximately 61KD (p61BRAF(V600E)). p61BRAF(V600E) exhibited enhanced dimerization as compared to full length BRAF(V600E) in cells with low levels of RAS-GTP. Ectopic expression of p61BRAF(V600E) conferred resistance to the RAF inhibitor. Moreover, a mutation that disrupts dimerization of p61BRAF(V600E) restored its sensitivity to vemurafenib. In tumors from patients that relapsed on vemurafenib we identified various splicing variants of BRAF(V600E), all of them lacking the RAS-binding domain (6/19). Disease progression samples obtained from a mutually exclusive subset (4/19) of the same group of patients harbored activating mutation in NRAS. We report the first RAF-inhibitor resistance mechanism that involves a structural change in BRAF and the first kinase inhibitor resistance mechanism that involves expression of aberrant splicing variants of the drug target. Tumors resistant to RAF inhibitors resulting from increased RAF dimerization retain sensitivity to inhibitors of downstream effectors of RAF such as MEK. Therefore, MEK inhibitors, if used in combination with RAF inhibitors, may delay or prevent the onset of this mechanism of resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-428. doi:1538-7445.AM2012-LB-428

Abstract 3562: Loss of the PTEN and RB1 tumor suppressors attenuates RAF-dependence in melanomas

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Oncogenic BRAF mutations are found in ∼8% of human tumors, with the highest frequency observed in melanoma (40-70%). BRAF mutations are common in nevi suggesting that BRAF mutation alone is insufficient for tumorigenesis. To identify concurrent mutations that condition BRAF/MEK dependence in V600E BRAF melanoma, we genotyped a panel of 149 melanoma cell lines. Seventy-eight cell lines (52%) harbored BRAF mutations with 72 of which are V600E mutants. Proteomic profiling of 41 out of the 72 V600E BRAF mutant cell lines demonstrated significant variability in the expression of PTEN and pAKT. Nine cell lines harboring V600E BRAF mutations express no PTEN protein and high pAKT by immunoblotting. All of the nine cell lines harbored small (1-2) base-pair insertions or deletions, missense mutations or focal deletions encompassing the PTEN locus. We have previously reported that mutations of BRAF are associated with enhanced and selective sensitivity to MEK inhibition when compared to either receptor tyrosine kinase-driven cells or cells harboring a RAS mutation. To determine the MAPK pathway dependence of V600E BRAF mutant cell lines as a function of PTEN expression, we used PD0325901, a selective allosteric inhibitor of MEK1/2. All V600E BRAF mutant, PTEN expressing cell lines were sensitive to PD0325901. All but two of the V600E BRAF mutant, PTEN null cells were also dependent upon MEK for proliferation. These data suggest that PTEN loss is insufficient to confer resistance to MAPK-pathway inhibition. Examination of the two models which displayed complete resistance to MEK inhibition (IC50 > 3,000 nM) revealed the presence of concurrent loss of PTEN and RB1. Inactivation of Rb functions by expressing human papillomavirus E7 protein in three BRAF mutant, PTEN-null models led to failure of the cells to arrest in G1 and complete resistance to MEK-inhibition. Moreover, RB1 alterations were mutually exclusive with loss of p16INK4A, suggesting that whereas p16INK4A and RB1 may have overlapping roles in preventing tumor formation, tumors with functional loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. V600EBRAF/RB-null/PTEN-null models were also resistant to the selective RAF inhibitor PLX4720. Consistent with these in vitro results, V600EBRAF/RB-null/PTEN-null xenografts were resistant to RAF and MEK inhibition whereas V600EBRAF/WTRB/PTEN-null cells exhibited an intermediate phenotype. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the MAP kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3562. doi:10.1158/1538-7445.AM2011-3562

Abstract LB-419: An in-frame deletion in the N-terminal regulatory domain of BRAF(V600E) causes resistance to the RAF inhibitor PLX4032

Drugs that compete with ATP for binding to RAF selectively inhibit ERK signaling in tumor cells with BRAF(V600E) mutation. In BRAF wild-type cells, these compounds induce ERK signaling. RAF family members form dimers in a RAS-dependent manner. In BRAF wild-type cells, binding of RAF inhibitors to one member of the dimer transactivates the other, non-bound member. In contrast, in tumors with BRAF(V600E) mutation, RAS activity is too low to support dimer formation and the drug thus inhibits ERK signaling. This property of RAF inhibitors such as PLX4032 is believed to account for the wide therapeutic index of this drug and its remarkable clinical activity in patients with BRAF(V600E) melanomas. However, despite the initial effectiveness of PLX4032, resistance invariably develops. The dimerization model predicts that induction of RAS activity in the tumor will confer resistance. RAS mutation and activation of receptor tyrosine kinases (RTK) including PDGFRs and IGF1R have been implicated, as has induction of expression of the COT kinase, which drives RAF-independent ERK signaling. In order to identify novel mechanisms of RAF-inhibitor resistance, we generated PLX4032-resistant cell lines by prolonged exposure of sensitive BRAF mutant melanoma cells to the RAF inhibitor PLX4032. Analysis revealed that resistance of these clones was associated with failure of the drug to inhibit ERK signaling. PLX4032-resistant cells remained dependent on ERK signaling, as growth was inhibited by a MEK inhibitor, albeit at slightly higher doses. We did not detect RAS mutations, upregulation of receptor tyrosine kinases (RTK), or of other MEK kinases (CRAF, COT, Mos) in these cells. In a subset of resistant clones, we identified the expression of a variant form of BRAF(V600E), with an in-frame deletion within the N-terminal regulatory domain and a molecular weight of about 61KD (p61BRAF(V600E)). The N-terminal domain of RAF has been shown to negatively regulate the C-terminal catalytic domain, at least in part, by preventing dimerization. When expressed in 293H cells with intrincically low levels of RAS.GTP, dimerization of p61BRAF(V600E) is elevated compared to that of full-length BRAF(V600E). Expression of p61BRAF(V600E) in 293H cells rendered ERK signaling insensitive to inhibition by PLX4032, whereas a mutation in the dimerization domain of p61BRAF(V600E) restored sensitivity. Thus, in addition to BRAF gatekeeper mutations, there are at least three potential mechanisms that confer resistance by blunting the inhibition of RAF kinase by the inhibitor, all of which promote RAF dimerization: increased RAS.GTP due to RAS mutation or upstream (RTK) activation, RAF overexpression and deletion of the RAS binding domain within the N-terminus of RAF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-419. doi:10.1158/1538-7445.AM2011-LB-419

Abstract LB-190: RAF inhibitors transactivate RAF dimers and ERK signaling in a wild-type BRAF-dependent manner

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Discussion Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-190.

Abstract 1971: Concurrent loss of PTEN and RB is sufficient to confer BRAF independence in melanomas harboring V600E BRAF mutations

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BRAF mutations are found in ∼8% of human tumors, with the highest frequency observed in melanoma (40-70%). Supporting its classification as an oncogene, V600E BRAF stimulates ERK signaling, induces proliferation and is capable in model systems of promoting transformation. BRAF mutations are, however, common in nevi suggesting that BRAF mutation alone is insufficient for tumorigenesis. With the goal of identifying concurrent mutations that condition BRAF/MEK dependence in V600E BRAF melanoma, we performed MALDI-TOF mass spectrometry genotyping on a panel of 149 melanoma cell lines. Seventy-nine (53%) harbored BRAF mutations. Proteomic profiling of the class of V600E BRAF mutant cell lines demonstrated significant variability in the expression of PTEN and pAKT. Nine cells lines harboring V600E BRAF mutations were found to express no PTEN protein and high pAKT by immunoblotting. The majority of these cell lines had small (1-2) base-pair insertions or deletions leading to a frame shift and early truncation. Array CGH confirmed focal homozygous deletion of the PTEN gene in three of the models. These deletions were small and in one case detected by the Agilent 1M but not the 244K platform. We have previously reported that mutations of BRAF are associated with enhanced and selective sensitivity to MEK inhibition when compared to either receptor tyrosine kinase-driven cells or cells harboring a RAS mutation. To determine the MAPK pathway dependence of V600E BRAF mutant cell lines as a function of PTEN expression, we used PD0325901, a selective allosteric inhibitor of MEK1/2. All V600E BRAF mutant, PTEN expressing cell lines were sensitive to PD0325901. All but two of the V600E BRAF mutant, PTEN null cells were also dependent upon MEK for proliferation with IC50 values ranging from 1.1 to >500nM. These data suggest that PTEN loss is insufficient to confer resistance to MAPK-pathway inhibition. Further characterization of the two models which displayed complete resistance to MEK inhibition revealed that both harbored concurrent loss of PTEN and RB. These models were also resistant to the selective RAF inhibitor PLX4720. To determine whether loss of RB function was sufficient to confer RAF/MEK-independence in a BRAF mutant, PTEN-null context, we stably infected viral E7 into three MEK-dependent, BRAF mutant, PTEN null models. Infection of E7 led to resistance to MEK-inhibition in all three models. These data suggest that no single mutational event is likely associated with de novo resistance to MEK-inhibition in V600E BRAF mutant melanoma. Loss of PTEN expression and activation of AKT may however be associated with diminished responsiveness to RAF/MEK-inhibition. Further, tumors with concurrent PTEN and RB loss may represent a subset of BRAF mutant patients who derive no clinical benefit from inhibitors of the MAPK pathway such as PLX4032. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1971.