Tumor regression and resistance mechanisms upon CDK4 and RAF1 inactivation in KRAS/P53 mutant lung adenocarcinomas.

KRAS mutant lung adenocarcinomas remain intractable for targeted therapies. Genetic interrogation of KRAS downstream effectors, including the MAPK pathway and the interphase CDKs, identified CDK4 and RAF1 as the only targets whose genetic inactivation induces therapeutic responses without causing unacceptable toxicities. Concomitant CDK4 inactivation and RAF1 ablation prevented tumor progression and induced complete regression in 25% of KRAS/p53-driven advanced lung tumors, yet a significant percentage of those tumors that underwent partial regression retained a population of CDK4/RAF1-resistant cells. Characterization of these cells revealed two independent resistance mechanisms implicating hypermethylation of several tumor suppressors and increased PI3K activity. Importantly, these CDK4/RAF1-resistant cells can be pharmacologically controlled. These studies open the door to new therapeutic strategies to treat KRAS mutant lung cancer, including resistant tumors.

Antileukemia activity of the novel peptidic CXCR4 antagonist LY2510924 as monotherapy and in combination with chemotherapy.

Targeting the stromal cell-derived factor 1α (SDF-1α)/C-X-C chemokine receptor type 4 (CXCR4) axis has been shown to be a promising therapeutic approach to overcome chemoresistance in acute myeloid leukemia (AML). We investigated the antileukemia efficacy of a novel peptidic CXCR4 antagonist, LY2510924, in preclinical models of AML. LY2510924 rapidly and durably blocked surface CXCR4 and inhibited stromal cell-derived factor 1 (SDF-1)α-induced chemotaxis and prosurvival signals of AML cells at nanomolar concentrations more effectively than the small-molecule CXCR4 antagonist AMD3100. In vitro, LY2510924 chiefly inhibited the proliferation of AML cells with little induction of cell death and reduced protection against chemotherapy by stromal cells. In mice with established AML, LY2510924 caused initial mobilization of leukemic cells into the circulation followed by reduction in total tumor burden. LY2510924 had antileukemia effects as monotherapy as well as in combination with chemotherapy. Gene expression profiling of AML cells isolated from LY2510924-treated mice demonstrated changes consistent with loss of SDF-1α/CXCR4 signaling and suggested reduced proliferation and induction of differentiation, which was proved by showing the attenuation of multiple prosurvival pathways such as PI3K/AKT, MAPK, and ß-catenin and myeloid differentiation in vivo. Effective disruption of the SDF-1α/CXCR4 axis by LY2510924 may translate into effective antileukemia therapy in future clinical applications.

Reactivation of mitogen-activated protein kinase (MAPK) pathway by FGF receptor 3 (FGFR3)/Ras mediates resistance to vemurafenib in human B-RAF V600E mutant melanoma.

Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma.

Safety and Pharmacokinetics of CXCR4 Peptide Antagonist, LY2510924, in Combination with Durvalumab in Advanced Refractory Solid Tumors.

Purpose: This was an open-label phase 1a study assessing the maximum tolerated dose (MTD), safety, and tolerability of CXCR4 peptide antagonist, LY2510924, administered in combination with durvalumab in patients with advanced refractory solid tumors. Methods: Patients received LY2510924 at 20, 30, or 40 mg subcutaneous (SC) once daily in combination with durvalumab at 1500 mg intravenously (IV) on day 1 of each 28-day cycle. The primary objective was to assess the MTD and safety of LY2510924 SC daily in combination with durvalumab in patients with advanced (metastatic and/or unresectable) solid tumors. Secondary objectives included pharmacokinetics (PK) and the antitumor activity of LY2510924 in combination with durvalumab. Exploratory objectives were biomarker analysis, including pharmacodynamic markers, relevant to LY2510924 and durvalumab, including immune functioning, drug targets, cancer-related pathways, and the disease state. Results: Nine patients (three each at 20, 30, and 40 mg) were enrolled in the study (eight patients with pancreatic cancer and one patient with rectal cancer). The majority of patients completed one or two cycles (100.0% ≥ 1 cycle; 88.9% ≥ 2 cycles) of LY2510924 and durvalumab. No dose limiting toxicities were reported. Most common (>10%) treatment-emergent adverse events were injection-site reaction (44.4%), fatigue (33.3%), and increased white blood cell count (33.3%). PK parameters for combination were similar to those reported in previous studies when given as monotherapy. Best overall response of stable disease was observed in four (44.4%) patients and one patient had unconfirmed partial response. Conclusion: The recommended phase 2 dose is 40 mg SC once-daily LY2510924 in combination with durvalumab 1500 mg IV and showed acceptable safety and tolerability in patients with advanced refractory tumors.

Identifying metastasis-initiating miRNA-target regulations of colorectal cancer from expressional changes in primary tumors.

Colorectal cancer (CRC) is prevalent with high mortality, with liver metastasis contributing as a major factor that worsens the survival of patients. The roles of miRNAs in CRC have been elucidated, subsequent to recent studies that suggest the involvement of miRNAs in cancer biology. In this study, we compare the miRNA and gene expression profiles of primary tumors between two groups of patients (with and without liver metastasis) to identify the metastasis-initiating microRNA-target gene regulations. Analysis from 33 patients with metastasis and 14 patients without metastasis revealed that 17 miRNAs and their 198 predicted target genes are differentially expressed, where the target genes showed association with cancer progression and metastasis with statistical significance. In order to evaluate the clinical implications of the findings, we classified CRC patients of independent data into two groups based on the identified miRNA-target regulations, where one group was closer to primary tumors with metastasis than the other group. The comparison of survival showed statistically significant difference, thereby implying the roles of the identified miRNA-target regulations in cancer progression and metastasis. The identification of metastasis-initiating miRNA-target regulations in this study will lead to better understanding of the roles of miRNAs in CRC progression.

Molecular dissection of CRC primary tumors and their matched liver metastases reveals critical role of immune microenvironment, EMT and angiogenesis in cancer metastasis.

Metastasis is the primary cause of cancer mortality. The primary tumors of colorectal cancer (CRC) often metastasize to the liver. In this study, we have collected 122 samples from 45 CRC patients. Among them, 32 patients have primary tumors, adjacent normal tissues, and matched liver metastases. Thirteen patients have primary tumors without distant metastasis and matched normal tissues. Characterization of these samples was conducted by whole-exome and RNA sequencing and SNP6.0 analysis. Our results revealed no significant difference in genetic alterations including common oncogenic mutations, whole genome mutations and copy number variations between primary and metastatic tumors. We then assembled gene co-expression networks and identified metastasis-correlated gene networks of immune-suppression, epithelial-mesenchymal transition (EMT) and angiogenesis as the key events and potentially synergistic drivers associated with CRC metastasis. Further independent cohort validation using published datasets has verified that these specific gene networks are up regulated throughout the tumor progression. The gene networks of EMT, angiogenesis, immune-suppression and T cell exhaustion are closely correlated with the poor patient outcome and intrinsic anti-PD-1 resistance. These results offer insights of combinational strategy for the treatment of metastatic CRC.

A Phase I Study of LY3009120, a Pan-RAF Inhibitor, in Patients with Advanced or Metastatic Cancer.

Mutations in ERK signaling drive a significant percentage of malignancies. LY3009120, a pan-RAF and dimer inhibitor, has preclinical activity in RAS- and BRAF-mutated cell lines including BRAF-mutant melanoma resistant to BRAF inhibitors. This multicenter, open-label, phase I clinical trial (NCT02014116) consisted of part A (dose escalation) and part B (dose confirmation) in patients with advanced/metastatic cancer. In part A, oral LY3009120 was dose escalated from 50 to 700 mg twice a day on a 28-day cycle. In part B, 300 mg LY3009120 was given twice a day. The primary objective was to identify a recommended phase II dose (RP2D). Secondary objectives were to evaluate safety, pharmacokinetics, and preliminary efficacy. Identification of pharmacodynamic biomarkers was exploratory. In parts A and B, 35 and 16 patients were treated, respectively (N = 51). In part A, 6 patients experienced eight dose-limiting toxicities. The RP2D was 300 mg twice a day. Common (>10%) any-grade drug-related treatment-emergent adverse events were fatigue (n = 15), nausea (n = 12), dermatitis acneiform (n = 10), decreased appetite (n = 7), and maculopapular rash (n = 7). The median duration of treatment was 4 weeks; 84% of patients completed one or two cycles of treatment. Exposures observed at 300 mg twice a day were above the preclinical concentration associated with tumor regression. Eight patients had a best overall response of stable disease; there were no complete or partial clinical responses. Despite adequate plasma exposure levels, predicted pharmacodynamic effects were not observed.

ERK Inhibitor LY3214996 Targets ERK Pathway-Driven Cancers: A Therapeutic Approach Toward Precision Medicine.

The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).

Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers.

We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.

Rapid and recurrent neutrophil mobilization regulated by T134, a CXCR4 peptide antagonist.

The CXCR4/stromal cell-derived factor-1 (SDF-1) axis plays important roles in development, leukocyte trafficking, HIV infection, and tumorigenesis. Its critical function in bone marrow stem cell and hematopoietic progenitor cell retention, homing and release has been well-characterized by genetic and pharmacological analyses. However, its role in neutrophil retention and release is still poorly understood. In this study, we demonstrated that T134, a peptide antagonist of human CXCR4, is also a potent antagonist of mouse CXCR4. Treatment of C57BL/6 mice with T134 resulted in a rapid and time-dependent increase of white blood cells (WBC) and neutrophils, as well as hematopoietic stem and progenitor cells in peripheral blood. Interestingly, recurrent WBC and neutrophil mobilization was achieved by repeated T134 treatment, and the T134-mediated increase and subsequent retreat of WBC and neutrophils correlated with T134 activity in the peripheral blood. Kinetic analysis revealed that T134 binding to CXCR4 did not induce any significant cell-surface receptor downregulation, indicating that T134-induced WBC and neutrophil mobilization is likely due to direct blockage of the CXCR4/SDF-1 interaction. The results from this study support an important role of CXCR4/SDF-1 axis in neutrophil retention and release in the marrow.

Chemical Proteomic Characterization of a Covalent KRASG12C Inhibitor.

The KRASG12C protein product is an attractive, yet challenging, target for small molecule inhibition. One option for therapeutic intervention is to design small molecule ligands capable of binding to and inactivating KRASG12C via formation of a covalent bond to the sulfhydryl group of cysteine 12. In order to better understand the cellular off-target interactions of Compound 1, a covalent KRASG12C inhibitor, we have completed a series of complementary chemical proteomics experiments in H358 cells. A new thiol reactive probe (TRP) was designed and used to construct a cellular target occupancy assay for KRASG12C. In addition, the thiol reactive probes allowed us to profile potential off-target interactions of Compound 1 with over 3200 cysteine residues. In order to complement the TRP data we designed Compound 2, an alkyne containing version of Compound 1, to serve as bait in competitive chemical proteomics experiments. Herein, we describe and compare data from both the TRP and the click chemistry probe pull down experiments.

Distinct mobilization of leukocytes and hematopoietic stem cells by CXCR4 peptide antagonist LY2510924 and monoclonal antibody LY2624587.

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy.

LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer.

Activating mutations in the KRAS and BRAF genes, leading to hyperactivation of the RAS/RAF/MAPK oncogenic signaling cascade, are common in patients with colorectal cancer (CRC). While selective BRAF inhibitors are efficacious in BRAFmut melanoma, they have limited efficacy in BRAFmut CRC patients. In a RASmut background, selective BRAF inhibitors are contraindicated due to paradoxical activation of the MAPK pathway through potentiation of CRAF kinase activity. A way to overcome such paradoxical activation is through concurrent inhibition of the kinase activity of both RAF isoforms. Here, we further examined the effects of LY3009120, a panRAF and RAF dimer inhibitor, in human models of CRC with various mutational backgrounds. We demonstrate that LY3009120 induced anti-proliferative effects in BRAFmut and KRASmut CRC cell lines through G1-cell cycle arrest. The anti-proliferative effects of LY3009120 in KRASmut CRC cell lines phenocopied molecular inhibition of RAF isoforms by simultaneous siRNA-mediated knockdown of ARAF, BRAF and CRAF. Additionally, LY3009120 displayed significant activity in in vivo BRAFmut and KRASmut CRC xenograft models. Examination of potential resistance to LY3009120 demonstrated RAF-independent ERK and AKT activation in the KRASmut CRC cell line HCT 116. These findings describe the preclinical activity of a panRAF inhibitor in a BRAFmut and KRASmut CRC setting.

Mouse PDX Trial Suggests Synergy of Concurrent Inhibition of RAF and EGFR in Colorectal Cancer with BRAF or KRAS Mutations.

Purpose: To evaluate the antitumor efficacy of cetuximab in combination with LSN3074753, an analog of LY3009120 and pan-RAF inhibitor in 79 colorectal cancer patient-derived xenograft (PDX) models.Experimental Design: Seventy-nine well-characterized colorectal cancer PDX models were employed to conduct a single mouse per treatment group (n = 1) trial.Results: Consistent with clinical results, cetuximab was efficacious in wild-type KRAS and BRAF PDX models, with an overall response rate of 6.3% and disease control rate (DCR) of 20.3%. LSN3074753 was active in a small subset of PDX models that harbored KRAS or BRAF mutations. However, the combination treatment displayed the enhanced antitumor activity with DCR of 35.4%. Statistical analysis revealed that BRAF and KRAS mutations were the best predictors of the combinatorial activity and were significantly associated with synergistic effect with a P value of 0.01 compared with cetuximab alone. In 12 models with BRAF mutations, the combination therapy resulted in a DCR of 41.7%, whereas either monotherapy had a DCR of 8.3%. Among 44 KRAS mutation models, cetuximab or LSN3074753 monotherapy resulted in a DCR of 13.6% or 11.4%, respectively, and the combination therapy increased DCR to 34.1%. Molecular analysis suggests that EGFR activation is a potential feedback and resistant mechanism of pan-RAF inhibition.Conclusions: MAPK and EGFR pathway activations are two major molecular hallmarks of colorectal cancer. This mouse PDX trial recapitulated clinical results of cetuximab. Concurrent EGFR and RAF inhibition demonstrated synergistic antitumor activity for colorectal cancer PDX models with a KRAS or BRAF mutation. Clin Cancer Res; 23(18); 5547-60. ©2017 AACR.

Identification and expression of novel isoforms of human stromal cell-derived factor 1.

Stromal Cell-derived factor 1 (SDF-1) is a CXC chemokine that binds to the CXCR4 receptor. Recent publication indicates that the SDF-1/CXCR4 signaling pathway plays a pivotal role during development and in many patho-physiological conditions including hematopoiesis, blood vessel formation, cancer metastasis, angiogenesis and HIV infection. Two human SDF-1 isoforms, SDF-1alpha and SDF-1beta, have been reported to date. Here we report the identification of four additional human SDF-1 isoforms derived from alternative splicing events, SDF-1gamma, SDF-1delta, SDF-1epsilon and SDF-1phi. These SDF-1 splice variants all share the same first three exons but contain different fourth exons. The human SDF-1 gene spans over 88 kilobase-pairs on chromosome 10. Using the semi-quantitative RT-PCR method, we determined the tissue distribution of these SDF-1 isoforms. SDF-1alpha and SDF-1beta share similar expression patterns and the highest expression were detected in liver, pancreas and spleen. SDF-1gamma seems to be the human orthologue of recently isolated rat SDF-1gamma, and its expression was only detected in the heart. SDF-1delta expression can be detected in several adult tissues but the highest expression was detected in fetal liver. When transfected into HEK293 cells, all the SDF-1 isoforms can be detected as secreted proteins in the cell culture media. The conditioned media from transfected cells can stimulate cell migration in a CXCR4-dependent manner. These data suggest that the novel SDF-1 splice variants encode functional proteins.

Akt activation, but not extracellular signal-regulated kinase activation, is required for SDF-1alpha/CXCR4-mediated migration of epitheloid carcinoma cells.

Emerging evidence shows that the stromal cell-derived factor 1 (SDF-1)/CXCR4 interaction regulates multiple cell signaling pathways and a variety of cellular functions such as cell migration, proliferation, and survival. There is little information linking the cellular functions and individual signaling pathways mediated by SDF-1 and CXCR4 in human cancer cells. In this study, we have shown that human epitheloid carcinoma HeLa cells express functional CXCR4 by reverse transcription-PCR, immunofluorescent staining, and 125I-SDF-1alpha ligand binding analyses. The treatment of HeLa cells with recombinant SDF-1alpha results in time-dependent Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) activations. The SDF-1alpha-induced Akt and ERK1/2 activations are CXCR4 dependent as confirmed by their total inhibition by T134, a CXCR4-specific peptide antagonist. Cell signaling analysis with pathway-specific inhibitors reveals that SDF-1alpha-induced Akt activation is not required for ERK1/2 activation and vice versa, indicating that activations of Akt and ERK1/2 occur independently. Functional analysis shows that SDF-1alpha induces a CXCR4-dependent migration of HeLa cells. The migration can be totally blocked by phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, whereas mitogen-activated protein/ERK kinase inhibitors, PD98059 and U0126, have no significant effect on SDF-1alpha-induced migration, suggesting that Akt activation, but not ERK1/2 activation, is required for SDF-1alpha-induced migration of epitheloid carcinoma cells.

Inhibition of CXCR4 by LY2624587, a Fully Humanized Anti-CXCR4 Antibody Induces Apoptosis of Hematologic Malignancies.

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.

Oncogenic BRAF Deletions That Function as Homodimers and Are Sensitive to Inhibition by RAF Dimer Inhibitor LY3009120.

UNLABELLED: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the ß3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.

Overexpression of G protein-coupled receptors in cancer cells: involvement in tumor progression.

G protein-coupled receptors (GPCRs) play important roles in a variety of biological and pathological processes. They are considered among the most desirable targets for drug development. Recent studies have demonstrated that many GPCRs, such as endothelin receptors, chemokine receptors and lysophosphatidic acid receptors have been implicated in the tumorigenesis and metastasis of multiple human cancers. In this study, we conducted an in silico analysis of GPCR gene expression in primary human tumors by analyzing some publicly available gene expression profiling data. Statistical analysis was performed on eight microarray data sets of non-small cell lung cancer, breast cancer, prostate cancer, melanoma, gastric cancer and diffused large B cell lymphoma to identify GPCRs that are up-regulated in primary or metastatic cancer cells. Our analysis has demonstrated overexpression of several GPCRs in primary tumor cells, including chemokine receptors and protease-activated receptors that were shown to be important for tumorigenesis by previous studies. In addition, we have uncovered several GPCRs, such as neuropeptide receptors, adenosine A2B receptor, P2Y purinoceptor, calcium-sensing receptor and metabotropic glutamate receptors, that are expressed at a significantly higher level in some cancer tissue and may play a role in cancer progression. Analysis of cancer samples in different disease stages also suggests that some GPCRs, such as endothelin receptor A, may be involved in early tumor progression and others, such as CXCR4, may play a critical role in tumor invasion and metastasis. The present study demonstrates the value of publicly available microarray data as a resource to gain more understanding of cancer biology, to validate previous findings from in vitro experiments, and to identify potential novel anticancer targets and biomarkers.

Co-targeting BRAF and cyclin dependent kinases 4/6 for BRAF mutant cancers.

Selective BRAF inhibitors have demonstrated significant clinical benefit in melanoma patients harboring oncogenic BRAF mutations. However, the majority of such patients either exhibit de novo resistance from the beginning of the treatment or acquire resistance and eventually relapse. Despite tremendous progress in understanding the underlying mechanisms of resistance, overcoming resistance to BRAF inhibitors remains an unmet medical need. Constitutive activation of cyclin-dependent kinases (CDK) 4/6 as a result of genetic aberrations including CDKN2A inactivation and CCND1 amplification is common across many cancer types and frequently co-occurs with oncogenic BRAF mutations. Also, cyclin D1 overexpression is a common feature of resistance to BRAF inhibitors. Here we review CDK4/6 as a therapeutic target in BRAF mutant cancers and discuss emerging evidence supporting a critical role of cyclin D1/CDK4/6 axis in de novo and acquired resistance to BRAF inhibitors. Co-targeting CDK4/6 and BRAF could be a more effective therapy to augment clinical response of BRAF inhibitors and overcome resistance in BRAF mutant cancers.