5384 Tumor-associated macrophages have been correlated with poor outcome in several types of canc... more 5384 Tumor-associated macrophages have been correlated with poor outcome in several types of cancer, although a cause-effect relation has not been established. The cytokine CSF-1 regulates the growth, differentiation, and activation of macrophages, and there is evidence in mouse models to support the hope that interference with CSF-1 signaling may lead to a benefit for cancer patients. CSF-1 is also a key regulator of osteoclast differentiation, so this benefit conceivably may extend to delay or prevent the effects of pain and bone resorption commonly observed in cancer patients reaching a metastatic stage involving bone. We have developed a series of small molecule compounds that inhibit the protein tyrosine kinase activity of FMS, the transmembrane receptor for CSF-1. Chemistry designs were iteratively guided by co-crystallography with the FMS catalytic domain, and a portfolio was obtained of compounds with high-affinity ( 100-fold). Biochemical activities were confirmed using cell-based assays, including an assay in which Ba/F3 pre-B cells were engineered to express a fusion protein having the N-terminus of BCR linked to the intracellular catalytic domain of FMS. Whereas the parent Ba/F3 cells require IL-3 for growth, these Ba/F3-FMS cells do not, and addition of the FMS inhibitors to the Ba/F3-FMS cultures caused apoptosis and death in the absence, but not presence, of added IL-3. When Ba/F3-FMS cells (5x106 cells) were introduced through the tail vein into female athymic nude mice, a splenomegaly was observed, with spleen weights increasing 20-fold from 0.06 g to 1.2 g after 14-17 days. Oral administration of the FMS inhibitors to the injected mice reduced the splenomegaly in a dose-dependent manner, indicating that the compounds have pharmacokinetic properties acceptable for reaching effective doses in animals. Dosing during only the first 3 days or the last 3 days reduced the splenomegaly partially, indicating that the growth of Ba/F3-FMS cells in the spleen is sensitive throughout the growth period to inhibition of the FMS kinase, suggesting this model is a useful mimic of longer-term inhibitor treatment regimens. The FMS inhibitors were also tested in an MCF-7 breast cancer xenograft model. MCF-7 cells were passed as tumor fragments from tumors established in nude mice, and no estradiol supplementation was given. After 4 weeks of treatment, the FMS inhibitor reduced the tumor volume by 40%. In an MDA-MB-231-LUC breast cancer metastasis model, in which cells were injected into the left heart ventricle, treatment with FMS inhibitor from days 28 to 34 after injection led to a 60% decrease in tumor burden in the bone. These initial studies are supportive of further tests of FMS as a target in breast cancer, and work on these compounds and newer derivatives are in progress.
Epigenetic changes in cancer are thought to contribute to regulation of tumor invasion and metast... more Epigenetic changes in cancer are thought to contribute to regulation of tumor invasion and metastasis, but this previously has not been studied at a genome wide level in melanoma. We analyzed the methylome of 44 cases of malignant melanoma with the HELP (HpaII tiny fragment enriched by LM-PCR) assay and compared it with healthy melanocyte controls. We observed widespread demethylation in malignant melanoma, preferentially outside of CpG islands. The epigenomic loss of methylation was independent of mutational status of BRAF, RAS and Kit. Comparison of primary and metastatic lesions demonstrated that demethylation occurs early during carcinogenesis with very few additional alterations in advanced tumors. Parallel transcriptomic analysis revealed many known and novel oncogenic pathways that were aberrantly expressed and regulated by loss of DNA methylation. Strikingly, the colony stimulating factor-1 receptor (CSF1R, c-fms) was aberrantly expressed and hypomethylated in nearly all cases. CSF1R is a transmembrane tyrosine kinase receptor that predominantly regulates macrophages, osteoclasts, and microglia, but is known to sometimes be aberrantly expressed by malignant cells in Hodgkins lymphoma. The expression of CSF1R on malignant melanocytes was validated by immunohistochemical analysis of primary tumors. In several melanoma cell lines (A2058, WM-266-4, SK-MEL-2, M14c#5) we found through PCR sequencing of the cDNA 5′ untranslated region that the CSF1R can be expressed through an aberrant promoter, as has been described for Hodgkin lymphoma. A custom Taqman assay was developed for this unique transcript, and then used to detect the transcript in 4 of 40 samples in a panel of melanoma biopsies, suggesting that aberrant CSF1R expression in melanoma is not uncommon. Expression of CSF1R protein in the cell lines was confirmed by FACS using anti-CD115 antibodies, and by Western blot using antibodies directed to the C-terminus. Expression of the ligand CSF-1 was also found in the melanoma cells by both ELISA and Taqman assays. Inhibition of in vitro cell growth by PLX3397, a clinically relevant small molecule inhibitor of CSF1R kinase, could be observed in 3D cell culture, indicating that under some conditions an autocrine stimulation of growth occurs. shRNA mediated knockdown of CSF1R also demonstrated decreased colony size and increased apoptosis in 3D culture conditions. The invasiveness of melanoma cells was decreased after treatment with PLX3397 or anti-CSF1 antibodies, suggesting a role for melanoma cancer cell expression of CSF1R in metastasis. Since three of cell lines possess an oncogenic BRAF mutation, co-inhibition of CSF1R and BRAF was tested and resulted in synergistic blockade of cell growth in vitro and A2058 xenograft growth in vivo. The CSF1R inhibitor, PLX3397, is under investigation in clinical trials for breast, glioma, and other cancers, and these data present a preclinical rationale for its study in malignant melanoma. This abstract is also being presented as Poster A06. Citation Format: Orsolya Giricz, Yongkai Mo, Caroline Hu, Kimberly Dahlman, Sanchari Bhattacharyya, Hoa Nguyen, Bernice Matusow, Tushar Bhagat, Yiting Yu, Rafe Shellooe, Elizabeth Burton, Gaston Habets, John Greally, Kenny Paraic, Jeffrey Sosman, Gideon Bollag, Brian West, Amit Verma. Integrated epigenomic profiling reveals widespread demethylation in melanoma and reveals CSF-1 Receptor as an aberrant regulator of malignant growth and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR06.
Supplementary Table 1. Crystallographic Data and Refinement statistics. Supplementary Table 2. Co... more Supplementary Table 1. Crystallographic Data and Refinement statistics. Supplementary Table 2. Comparing Target Inhibition by Quizartinib and PLX3397 in Engineered Ba/F3 cells Expressing FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y. Supplementary Table 3. Inhibitory Concentration (IC50) for Proliferation of Human Leukemia Cell Lines in PLX3397. Supplementary Table 4. 48 Hour Inhibitory Concentration (IC50) for Proliferation of Ba/F3 Cells Expressing Quizartinib and PLX3397-Resistant FLT3-ITD Mutant Isoforms. Supplementary Table 5. Patient Characteristics. Supplementary Table 6. Low Frequency FLT3 Kinase Domain Mutations of Uncertain Significance Observed at the Time of Resistance in Patient 1.14. Supplementary Figure 1. Composite omit map contoured at 1sigma level for the bound quizartinib and a structural water molecule. Supplementary Figure 2. Comparing the actual and previously predicted binding modes of quizartinib. Supplementary Figure 3. Structural model showing that D835 induc...
5384 Tumor-associated macrophages have been correlated with poor outcome in several types of canc... more 5384 Tumor-associated macrophages have been correlated with poor outcome in several types of cancer, although a cause-effect relation has not been established. The cytokine CSF-1 regulates the growth, differentiation, and activation of macrophages, and there is evidence in mouse models to support the hope that interference with CSF-1 signaling may lead to a benefit for cancer patients. CSF-1 is also a key regulator of osteoclast differentiation, so this benefit conceivably may extend to delay or prevent the effects of pain and bone resorption commonly observed in cancer patients reaching a metastatic stage involving bone. We have developed a series of small molecule compounds that inhibit the protein tyrosine kinase activity of FMS, the transmembrane receptor for CSF-1. Chemistry designs were iteratively guided by co-crystallography with the FMS catalytic domain, and a portfolio was obtained of compounds with high-affinity ( 100-fold). Biochemical activities were confirmed using c...
Importance Many cancer subtypes, including KIT-mutant gastrointestinal stromal tumors (GISTs), ar... more Importance Many cancer subtypes, including KIT-mutant gastrointestinal stromal tumors (GISTs), are driven by activating mutations in tyrosine kinases and may initially respond to kinase inhibitors but frequently relapse owing to outgrowth of heterogeneous subclones with resistance mutations. KIT inhibitors commonly used to treat GIST (eg, imatinib and sunitinib) are inactive-state (type II) inhibitors. Objective To assess whether combining a type II KIT inhibitor with a conformation-complementary, active-state (type I) KIT inhibitor is associated with broad mutation coverage and global disease control. Design, Setting, and Participants A highly selective type I inhibitor of KIT, PLX9486, was tested in a 2-part phase 1b/2a trial. Part 1 (dose escalation) evaluated PLX9486 monotherapy in patients with solid tumors. Part 2e (extension) evaluated PLX9486-sunitinib combination in patients with GIST. Patients were enrolled from March 2015 through February 2019; data analysis was performed from May 2020 through July 2020. Interventions Participants received 250, 350, 500, and 1000 mg of PLX9486 alone (part 1) or 500 and 1000 mg of PLX9486 together with 25 or 37.5 mg of sunitinib (part 2e) continuously in 28-day dosing cycles until disease progression, treatment discontinuation, or withdrawal. Main Outcomes and Measures Pharmacokinetics, safety, and tumor responses were assessed. Clinical efficacy end points (progression-free survival and clinical benefit rate) were supplemented with longitudinal monitoring of KIT mutations in circulating tumor DNA. Results A total of 39 PLX9486-naive patients (median age, 57 years [range, 39-79 years]; 22 men [56.4%]; 35 [89.7%] with refractory GIST) were enrolled in the dose escalation and extension parts. The recommended phase 2 dose of PLX9486 was 1000 mg daily. At this dose, PLX9486 could be safely combined with 25 or 37.5 mg daily of sunitinib continuously. Patients with GIST who received PLX9486 at a dose of 500 mg or less, at the recommended phase 2 dose, and with sunitinib had median (95% CI) progression-free survivals of 1.74 (1.54-1.84), 5.75 (0.99-11.0), and 12.1 (1.34-NA) months and clinical benefit rates (95% CI) of 14% (0%-58%), 50% (21%-79%), and 80% (52%-96%), respectively. Conclusions and Relevance In this phase 1b/2a nonrandomized clinical trial, type I and type II KIT inhibitors PLX9486 and sunitinib were safely coadministered at the recommended dose of both single agents in patients with refractory GIST. Results suggest that cotargeting 2 complementary conformational states of the same kinase was associated with clinical benefit with an acceptable safety profile. Trial Registration ClinicalTrials.gov Identifier: NCT02401815.
2583Background: First-generation BRAF inhibitors (BRAFi) show high response rates and prolonged s... more 2583Background: First-generation BRAF inhibitors (BRAFi) show high response rates and prolonged survival in some BRAFV600-mutant cancers; however, paradoxical activation of the RAF/MEK/ERK pathway ...
Epigenetic changes in cancer are thought to contribute to the regulation of invasion and metastas... more Epigenetic changes in cancer are thought to contribute to the regulation of invasion and metastasis. To study this at a genome-wide level in melanoma, we analyzed the methylome of 44 cases of malignant melanoma. We saw widespread demethylation occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions showed demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. The colony stimulating factor-1 receptor was aberrantly expressed and hypomethylated in nearly all cases. Its expression was validated by IHC and RNA-FISH on primary tumors and by qPCR, Western blotting and FACS in BRAF mutant and WT cell lines. CSF1R can be aberrantly expressed via an upstream LTR element in Hodgkin's lymphoma. After analyzing our patient samples and cell lines, we have found this aberrant transcript may be the dominant form in melanoma as well. Expression of one of its ligands IL34 was also shown in the cell lines by both ...
Resistance to current therapies still impacts a significant number of melanoma patients and can b... more Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition o...
Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in can... more Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is over-expressed in CLL and is enriched proximal to genes up-regulated or de novo expressed in CLL with known function in disease pathogenesis and progression. These genes, including key members of the BCR signaling pathway, provide rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel in vitro and in vivo pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in pre-clinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of ...
Epigenetic changes in cancer are thought to contribute to regulation of invasion and metastasis. ... more Epigenetic changes in cancer are thought to contribute to regulation of invasion and metastasis. To study this at a genome-wide level in melanoma we analyzed the methylome of 44 cases of malignant melanoma. We saw widespread demethylation in melanoma occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions showed demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. The colony stimulating factor-1 receptor was aberrantly expressed and hypomethylated in nearly all cases. The expression of CSF1R was validated by IHC on primary tumors and by qPCR and Western blotting in BRAF mutant and WT cell lines. CSF1R can be aberrantly expressed via an upstream LTR element in Hodgkin’s lymphoma. After analyzing our patient samples and the cell lines, we have found this aberrant transcript may be the dominant form in melanoma as well. Expression of one of its ligands IL34 was also shown in the cell lines by both ELI...
Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mito... more Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed…
5384 Tumor-associated macrophages have been correlated with poor outcome in several types of canc... more 5384 Tumor-associated macrophages have been correlated with poor outcome in several types of cancer, although a cause-effect relation has not been established. The cytokine CSF-1 regulates the growth, differentiation, and activation of macrophages, and there is evidence in mouse models to support the hope that interference with CSF-1 signaling may lead to a benefit for cancer patients. CSF-1 is also a key regulator of osteoclast differentiation, so this benefit conceivably may extend to delay or prevent the effects of pain and bone resorption commonly observed in cancer patients reaching a metastatic stage involving bone. We have developed a series of small molecule compounds that inhibit the protein tyrosine kinase activity of FMS, the transmembrane receptor for CSF-1. Chemistry designs were iteratively guided by co-crystallography with the FMS catalytic domain, and a portfolio was obtained of compounds with high-affinity ( 100-fold). Biochemical activities were confirmed using cell-based assays, including an assay in which Ba/F3 pre-B cells were engineered to express a fusion protein having the N-terminus of BCR linked to the intracellular catalytic domain of FMS. Whereas the parent Ba/F3 cells require IL-3 for growth, these Ba/F3-FMS cells do not, and addition of the FMS inhibitors to the Ba/F3-FMS cultures caused apoptosis and death in the absence, but not presence, of added IL-3. When Ba/F3-FMS cells (5x106 cells) were introduced through the tail vein into female athymic nude mice, a splenomegaly was observed, with spleen weights increasing 20-fold from 0.06 g to 1.2 g after 14-17 days. Oral administration of the FMS inhibitors to the injected mice reduced the splenomegaly in a dose-dependent manner, indicating that the compounds have pharmacokinetic properties acceptable for reaching effective doses in animals. Dosing during only the first 3 days or the last 3 days reduced the splenomegaly partially, indicating that the growth of Ba/F3-FMS cells in the spleen is sensitive throughout the growth period to inhibition of the FMS kinase, suggesting this model is a useful mimic of longer-term inhibitor treatment regimens. The FMS inhibitors were also tested in an MCF-7 breast cancer xenograft model. MCF-7 cells were passed as tumor fragments from tumors established in nude mice, and no estradiol supplementation was given. After 4 weeks of treatment, the FMS inhibitor reduced the tumor volume by 40%. In an MDA-MB-231-LUC breast cancer metastasis model, in which cells were injected into the left heart ventricle, treatment with FMS inhibitor from days 28 to 34 after injection led to a 60% decrease in tumor burden in the bone. These initial studies are supportive of further tests of FMS as a target in breast cancer, and work on these compounds and newer derivatives are in progress.
Epigenetic changes in cancer are thought to contribute to regulation of tumor invasion and metast... more Epigenetic changes in cancer are thought to contribute to regulation of tumor invasion and metastasis, but this previously has not been studied at a genome wide level in melanoma. We analyzed the methylome of 44 cases of malignant melanoma with the HELP (HpaII tiny fragment enriched by LM-PCR) assay and compared it with healthy melanocyte controls. We observed widespread demethylation in malignant melanoma, preferentially outside of CpG islands. The epigenomic loss of methylation was independent of mutational status of BRAF, RAS and Kit. Comparison of primary and metastatic lesions demonstrated that demethylation occurs early during carcinogenesis with very few additional alterations in advanced tumors. Parallel transcriptomic analysis revealed many known and novel oncogenic pathways that were aberrantly expressed and regulated by loss of DNA methylation. Strikingly, the colony stimulating factor-1 receptor (CSF1R, c-fms) was aberrantly expressed and hypomethylated in nearly all cases. CSF1R is a transmembrane tyrosine kinase receptor that predominantly regulates macrophages, osteoclasts, and microglia, but is known to sometimes be aberrantly expressed by malignant cells in Hodgkins lymphoma. The expression of CSF1R on malignant melanocytes was validated by immunohistochemical analysis of primary tumors. In several melanoma cell lines (A2058, WM-266-4, SK-MEL-2, M14c#5) we found through PCR sequencing of the cDNA 5′ untranslated region that the CSF1R can be expressed through an aberrant promoter, as has been described for Hodgkin lymphoma. A custom Taqman assay was developed for this unique transcript, and then used to detect the transcript in 4 of 40 samples in a panel of melanoma biopsies, suggesting that aberrant CSF1R expression in melanoma is not uncommon. Expression of CSF1R protein in the cell lines was confirmed by FACS using anti-CD115 antibodies, and by Western blot using antibodies directed to the C-terminus. Expression of the ligand CSF-1 was also found in the melanoma cells by both ELISA and Taqman assays. Inhibition of in vitro cell growth by PLX3397, a clinically relevant small molecule inhibitor of CSF1R kinase, could be observed in 3D cell culture, indicating that under some conditions an autocrine stimulation of growth occurs. shRNA mediated knockdown of CSF1R also demonstrated decreased colony size and increased apoptosis in 3D culture conditions. The invasiveness of melanoma cells was decreased after treatment with PLX3397 or anti-CSF1 antibodies, suggesting a role for melanoma cancer cell expression of CSF1R in metastasis. Since three of cell lines possess an oncogenic BRAF mutation, co-inhibition of CSF1R and BRAF was tested and resulted in synergistic blockade of cell growth in vitro and A2058 xenograft growth in vivo. The CSF1R inhibitor, PLX3397, is under investigation in clinical trials for breast, glioma, and other cancers, and these data present a preclinical rationale for its study in malignant melanoma. This abstract is also being presented as Poster A06. Citation Format: Orsolya Giricz, Yongkai Mo, Caroline Hu, Kimberly Dahlman, Sanchari Bhattacharyya, Hoa Nguyen, Bernice Matusow, Tushar Bhagat, Yiting Yu, Rafe Shellooe, Elizabeth Burton, Gaston Habets, John Greally, Kenny Paraic, Jeffrey Sosman, Gideon Bollag, Brian West, Amit Verma. Integrated epigenomic profiling reveals widespread demethylation in melanoma and reveals CSF-1 Receptor as an aberrant regulator of malignant growth and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR06.
Supplementary Table 1. Crystallographic Data and Refinement statistics. Supplementary Table 2. Co... more Supplementary Table 1. Crystallographic Data and Refinement statistics. Supplementary Table 2. Comparing Target Inhibition by Quizartinib and PLX3397 in Engineered Ba/F3 cells Expressing FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y. Supplementary Table 3. Inhibitory Concentration (IC50) for Proliferation of Human Leukemia Cell Lines in PLX3397. Supplementary Table 4. 48 Hour Inhibitory Concentration (IC50) for Proliferation of Ba/F3 Cells Expressing Quizartinib and PLX3397-Resistant FLT3-ITD Mutant Isoforms. Supplementary Table 5. Patient Characteristics. Supplementary Table 6. Low Frequency FLT3 Kinase Domain Mutations of Uncertain Significance Observed at the Time of Resistance in Patient 1.14. Supplementary Figure 1. Composite omit map contoured at 1sigma level for the bound quizartinib and a structural water molecule. Supplementary Figure 2. Comparing the actual and previously predicted binding modes of quizartinib. Supplementary Figure 3. Structural model showing that D835 induc...
5384 Tumor-associated macrophages have been correlated with poor outcome in several types of canc... more 5384 Tumor-associated macrophages have been correlated with poor outcome in several types of cancer, although a cause-effect relation has not been established. The cytokine CSF-1 regulates the growth, differentiation, and activation of macrophages, and there is evidence in mouse models to support the hope that interference with CSF-1 signaling may lead to a benefit for cancer patients. CSF-1 is also a key regulator of osteoclast differentiation, so this benefit conceivably may extend to delay or prevent the effects of pain and bone resorption commonly observed in cancer patients reaching a metastatic stage involving bone. We have developed a series of small molecule compounds that inhibit the protein tyrosine kinase activity of FMS, the transmembrane receptor for CSF-1. Chemistry designs were iteratively guided by co-crystallography with the FMS catalytic domain, and a portfolio was obtained of compounds with high-affinity ( 100-fold). Biochemical activities were confirmed using c...
Importance Many cancer subtypes, including KIT-mutant gastrointestinal stromal tumors (GISTs), ar... more Importance Many cancer subtypes, including KIT-mutant gastrointestinal stromal tumors (GISTs), are driven by activating mutations in tyrosine kinases and may initially respond to kinase inhibitors but frequently relapse owing to outgrowth of heterogeneous subclones with resistance mutations. KIT inhibitors commonly used to treat GIST (eg, imatinib and sunitinib) are inactive-state (type II) inhibitors. Objective To assess whether combining a type II KIT inhibitor with a conformation-complementary, active-state (type I) KIT inhibitor is associated with broad mutation coverage and global disease control. Design, Setting, and Participants A highly selective type I inhibitor of KIT, PLX9486, was tested in a 2-part phase 1b/2a trial. Part 1 (dose escalation) evaluated PLX9486 monotherapy in patients with solid tumors. Part 2e (extension) evaluated PLX9486-sunitinib combination in patients with GIST. Patients were enrolled from March 2015 through February 2019; data analysis was performed from May 2020 through July 2020. Interventions Participants received 250, 350, 500, and 1000 mg of PLX9486 alone (part 1) or 500 and 1000 mg of PLX9486 together with 25 or 37.5 mg of sunitinib (part 2e) continuously in 28-day dosing cycles until disease progression, treatment discontinuation, or withdrawal. Main Outcomes and Measures Pharmacokinetics, safety, and tumor responses were assessed. Clinical efficacy end points (progression-free survival and clinical benefit rate) were supplemented with longitudinal monitoring of KIT mutations in circulating tumor DNA. Results A total of 39 PLX9486-naive patients (median age, 57 years [range, 39-79 years]; 22 men [56.4%]; 35 [89.7%] with refractory GIST) were enrolled in the dose escalation and extension parts. The recommended phase 2 dose of PLX9486 was 1000 mg daily. At this dose, PLX9486 could be safely combined with 25 or 37.5 mg daily of sunitinib continuously. Patients with GIST who received PLX9486 at a dose of 500 mg or less, at the recommended phase 2 dose, and with sunitinib had median (95% CI) progression-free survivals of 1.74 (1.54-1.84), 5.75 (0.99-11.0), and 12.1 (1.34-NA) months and clinical benefit rates (95% CI) of 14% (0%-58%), 50% (21%-79%), and 80% (52%-96%), respectively. Conclusions and Relevance In this phase 1b/2a nonrandomized clinical trial, type I and type II KIT inhibitors PLX9486 and sunitinib were safely coadministered at the recommended dose of both single agents in patients with refractory GIST. Results suggest that cotargeting 2 complementary conformational states of the same kinase was associated with clinical benefit with an acceptable safety profile. Trial Registration ClinicalTrials.gov Identifier: NCT02401815.
2583Background: First-generation BRAF inhibitors (BRAFi) show high response rates and prolonged s... more 2583Background: First-generation BRAF inhibitors (BRAFi) show high response rates and prolonged survival in some BRAFV600-mutant cancers; however, paradoxical activation of the RAF/MEK/ERK pathway ...
Epigenetic changes in cancer are thought to contribute to the regulation of invasion and metastas... more Epigenetic changes in cancer are thought to contribute to the regulation of invasion and metastasis. To study this at a genome-wide level in melanoma, we analyzed the methylome of 44 cases of malignant melanoma. We saw widespread demethylation occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions showed demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. The colony stimulating factor-1 receptor was aberrantly expressed and hypomethylated in nearly all cases. Its expression was validated by IHC and RNA-FISH on primary tumors and by qPCR, Western blotting and FACS in BRAF mutant and WT cell lines. CSF1R can be aberrantly expressed via an upstream LTR element in Hodgkin's lymphoma. After analyzing our patient samples and cell lines, we have found this aberrant transcript may be the dominant form in melanoma as well. Expression of one of its ligands IL34 was also shown in the cell lines by both ...
Resistance to current therapies still impacts a significant number of melanoma patients and can b... more Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition o...
Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in can... more Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is over-expressed in CLL and is enriched proximal to genes up-regulated or de novo expressed in CLL with known function in disease pathogenesis and progression. These genes, including key members of the BCR signaling pathway, provide rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel in vitro and in vivo pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in pre-clinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of ...
Epigenetic changes in cancer are thought to contribute to regulation of invasion and metastasis. ... more Epigenetic changes in cancer are thought to contribute to regulation of invasion and metastasis. To study this at a genome-wide level in melanoma we analyzed the methylome of 44 cases of malignant melanoma. We saw widespread demethylation in melanoma occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions showed demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. The colony stimulating factor-1 receptor was aberrantly expressed and hypomethylated in nearly all cases. The expression of CSF1R was validated by IHC on primary tumors and by qPCR and Western blotting in BRAF mutant and WT cell lines. CSF1R can be aberrantly expressed via an upstream LTR element in Hodgkin’s lymphoma. After analyzing our patient samples and the cell lines, we have found this aberrant transcript may be the dominant form in melanoma as well. Expression of one of its ligands IL34 was also shown in the cell lines by both ELI...
Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mito... more Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed…
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