BET inhibition reforms the immune microenvironment and alleviates T cell dysfunction in chronic lymphocytic leukemia.

Redundant tumor microenvironment (TME) immunosuppressive mechanisms and epigenetic maintenance of terminal T cell exhaustion greatly hinder functional antitumor immune responses in chronic lymphocytic leukemia (CLL). Bromodomain and extraterminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T cell function and differentiation. Herein, we report that blocking BET protein function alleviates immunosuppressive networks in the CLL TME and repairs inherent CLL T cell defects. The pan-BET inhibitor OPN-51107 reduced exhaustion-associated cell signatures resulting in improved T cell proliferation and effector function in the Eµ-TCL1 splenic TME. Following BET inhibition (BET-i), TME T cells coexpressed significantly fewer inhibitory receptors (IRs) (e.g., PD-1, CD160, CD244, LAG3, VISTA). Complementary results were witnessed in primary CLL cultures, wherein OPN-51107 exerted proinflammatory effects on T cells, regardless of leukemic cell burden. BET-i additionally promotes a progenitor T cell phenotype through reduced expression of transcription factors that maintain terminal differentiation and increased expression of TCF-1, at least in part through altered chromatin accessibility. Moreover, direct T cell effects of BET-i were unmatched by common targeted therapies in CLL. This study demonstrates the immunomodulatory action of BET-i on CLL T cells and supports the inclusion of BET inhibitors in the management of CLL to alleviate terminal T cell dysfunction and potentially enhance tumoricidal T cell activity.

Phase IIa Study of PLX2853 in Gynecologic Cancers With Known ARID1A Mutation and Phase Ib/IIa Study of PLX2853/Carboplatin in Platinum-Resistant Epithelial Ovarian Cancer.

PURPOSE: The Bromodomain and Extra-Terminal (BET) domain proteins facilitate the development of many human cancers via epigenetic regulation. BET inhibitors may be effective in reversing platinum resistance in ovarian cancer (OC) and may generate synthetic lethality with ARID1A loss. PLX2853 is an orally active, small-molecule inhibitor of BET bromodomain-mediated interactions that exhibits low nanomolar potency in blocking all four BET family members. METHODS: We conducted a multicenter and open-label study with two parallel arms: a phase IIa study of PLX2853 monotherapy in patients with advanced gynecologic malignancies with an ARID1A mutation and a phase Ib/IIa combination study of PLX2853 plus carboplatin in women with platinum-resistant OC. The primary objectives were safety and tolerability for phase Ib and efficacy for both phase IIa portions. Thirty-four of 37 enrolled patients completed at least one post-baseline response assessment. RESULTS: Of the 14 evaluable patients on the monotherapy arm, 1 (7.1%) achieved a best overall response of partial response (PR), 5 (35.7%) had stable disease (SD), and 8 (57.1%) had progressive disease (PD). Of the 20 evaluable patients on the combination arm, 1 (5.0%) had PR, 9 (45.0%) had SD, and 10 (50%) had PD. CONCLUSION: This study confirmed the safety profile of PLX2853 and demonstrated the feasibility of combination with carboplatin. Although these results did not meet the prespecified response criteria, evidence of clinical activity highlights the rationale for further exploration of BET inhibitors in patients with ARID1A-mutated gynecologic malignancies, possibly in combination with agents targeting potential feedback mechanisms such as the PI3K pathway.

Structural and functional analyses of a germline KRAS T50I mutation provide insights into Raf activation.

A T50I substitution in the K-Ras interswitch domain causes Noonan syndrome and emerged as a third-site mutation that restored the in vivo transforming activity and constitutive MAPK pathway activation by an attenuated KrasG12D,E37G oncogene in a mouse leukemia model. Biochemical and crystallographic data suggested that K-RasT50I increases MAPK signal output through a non-GTPase mechanism, potentially by promoting asymmetric Ras:Ras interactions between T50 and E162. We generated a "switchable" system in which K-Ras mutant proteins expressed at physiologic levels supplant the fms like tyrosine kinase 3 (FLT3) dependency of MOLM-13 leukemia cells lacking endogenous KRAS and used this system to interrogate single or compound G12D, T50I, D154Q, and E162L mutations. These studies support a key role for the asymmetric lateral assembly of K-Ras in a plasma membrane-distal orientation that promotes the formation of active Ras:Raf complexes in a membrane-proximal conformation. Disease-causing mutations such as T50I are a valuable starting point for illuminating normal Ras function, elucidating mechanisms of disease, and identifying potential therapeutic opportunities for Rasopathy disorders and cancer.

Uterine leiomyosarcomas harboring MAP2K4 gene amplification are sensitive in vivo to PLX8725, a novel MAP2K4 inhibitor.

INTRODUCTION: Uterine leiomyosarcomas (uLMS) are rare, highly aggressive tumors. Up to 30% of uLMS may harbor gain of function (GOF) in the MAP2K4 gene, important for tumor cell proliferation, differentiation and metastasis. We investigated the in vivo activity of a novel MAP2K4 inhibitor, PLX8725, against uLMS harboring MAP2K4 gene-amplification. METHODS: Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or PLX8725 (50 mg/kg) were given via oral gavage daily on weekdays for up to 60 days. Tumor volume differences were calculated with two-way ANOVA. Pharmacokinetic (PK) and mechanistic studies of PLX8725 in uLMS PDX models were also performed. RESULTS: Both uLMS tumors evaluated demonstrated GOF in MAP2K4 (i.e., 3 CNV in both LEY-11 and LEY-16). Tumor growth inhibition was significantly greater in both PDX LEY-11 and PDX LEY-16 treated with PLX8725 when compared to controls (p < 0.001). Median overall survival was also significantly longer in both PDX LEY-11 (p = 0.0047) and PDX LEY-16 (p = 0.0058) treatment cohorts when compared to controls. PLX8725 oral treatment was well tolerated, and PK studies demonstrated that oral PLX8725 gives extended exposure in mice. Ex vivo tumor samples after PLX8725 exposure decreased phosphorylated-ATR, JNK and p38, and increased expression of apoptotic molecules on western blot. CONCLUSION: PLX8725 demonstrates promising in vivo activity against PDX models of uLMS harboring GOF alterations in the MAP2K4 gene with tolerable toxicity. Phase I trials of PLX8725 in advanced, recurrent, chemotherapy-resistant uLMS patients are warranted.

MEK inhibitors for neurofibromatosis type 1 manifestations: Clinical evidence and consensus.

The wide variety of clinical manifestations of the genetic syndrome neurofibromatosis type 1 (NF1) are driven by overactivation of the RAS pathway. Mitogen-activated protein kinase kinase inhibitors (MEKi) block downstream targets of RAS. The recent regulatory approvals of the MEKi selumetinib for inoperable symptomatic plexiform neurofibromas in children with NF1 have made it the first medical therapy approved for this indication in the United States, the European Union, and elsewhere. Several recently published and ongoing clinical trials have demonstrated that MEKi may have potential benefits for a variety of other NF1 manifestations, and there is broad interest in the field regarding the appropriate clinical use of these agents. In this review, we present the current evidence regarding the use of existing MEKi for a variety of NF1-related manifestations, including tumor (neurofibromas, malignant peripheral nerve sheath tumors, low-grade glioma, and juvenile myelomonocytic leukemia) and non-tumor (bone, pain, and neurocognitive) manifestations. We discuss the potential utility of MEKi in related genetic conditions characterized by overactivation of the RAS pathway (RASopathies). In addition, we review practical treatment considerations for the use of MEKi as well as provide consensus recommendations regarding their clinical use from a panel of experts.

Inhibition of Bromodomain and Extra Terminal (BET) Domain Activity Modulates the IL-23R/IL-17 Axis and Suppresses Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (GVHD) is the leading cause of non-relapse mortality following allogeneic hematopoietic cell transplantation. The majority of patients non-responsive to front line treatment with steroids have an estimated overall 2-year survival rate of only 10%. Bromodomain and extra-terminal domain (BET) proteins influence inflammatory gene transcription, and therefore represent a potential target to mitigate inflammation central to acute GVHD pathogenesis. Using potent and selective BET inhibitors Plexxikon-51107 and -2853 (PLX51107 and PLX2853), we show that BET inhibition significantly improves survival and reduces disease progression in murine models of acute GVHD without sacrificing the beneficial graft-versus-leukemia response. BET inhibition reduces T cell alloreactive proliferation, decreases inflammatory cytokine production, and impairs dendritic cell maturation both in vitro and in vivo. RNA sequencing studies in human T cells revealed that BET inhibition impacts inflammatory IL-17 and IL-12 gene expression signatures, and Chromatin Immunoprecipitation (ChIP)-sequencing revealed that BRD4 binds directly to the IL-23R gene locus. BET inhibition results in decreased IL-23R expression and function as demonstrated by decreased phosphorylation of STAT3 in response to IL-23 stimulation in human T cells in vitro as well as in mouse donor T cells in vivo. Furthermore, PLX2853 significantly reduced IL-23R+ and pathogenic CD4+ IFNγ+ IL-17+ double positive T cell infiltration in gastrointestinal tissues in an acute GVHD murine model. Our findings identify a role for BET proteins in regulating the IL-23R/STAT3/IL-17 pathway. Based on our preclinical data presented here, PLX51107 will enter clinical trial for refractory acute GVHD in a Phase 1 safety, biological efficacy trial.

Triple Therapy to Outwit the BRAF Oncogene.

BRAF-mutant cancers have been knocked down by BRAF and MEK inhibitor combos, yet the cancers always find a way to get back up. Adding a third medicine, namely, a dimer-selective BRAF inhibitor, can knock down longer and better.See related article by Adamopoulos et al., p. 1716.

Association of Combination of Conformation-Specific KIT Inhibitors With Clinical Benefit in Patients With Refractory Gastrointestinal Stromal Tumors: A Phase 1b/2a Nonrandomized Clinical Trial.

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.

Recurrent Mutations in Cyclin D3 Confer Clinical Resistance to FLT3 Inhibitors in Acute Myeloid Leukemia.

PURPOSE: Biomarkers of response and resistance to FLT3 tyrosine kinase inhibitors (TKI) are still emerging, and optimal clinical combinations remain unclear. The purpose of this study is to identify co-occurring mutations that influence clinical response to the novel FLT3 inhibitor pexidartinib (PLX3397). EXPERIMENTAL DESIGN: We performed targeted sequencing of pretreatment blasts from 29 patients with FLT3 internal tandem duplication (ITD) mutations treated on the phase I/II trial of pexidartinib in relapsed/refractory FLT3-ITD+ acute myeloid leukemia (AML). We sequenced 37 samples from 29 patients with available material, including 8 responders and 21 non-responders treated at or above the recommended phase II dose of 3,000 mg. RESULTS: Consistent with other studies, we identified mutations in NRAS, TP53, IDH2, and a variety of epigenetic and transcriptional regulators only in non-responders. Among the most frequently mutated genes in non-responders was Cyclin D3 (CCND3). A total of 3 individual mutations in CCND3 (Q276*, S264R, and T283A) were identified in 2 of 21 non-responders (one patient had both Q276* and S264R). No CCND3 mutations were found in pexidartinib responders. Expression of the Q276* and T283A mutations in FLT3-ITD MV4;11 cells conferred resistance to apoptosis, decreased cell-cycle arrest, and increased proliferation in the presence of pexidartinib and other FLT3 inhibitors. Inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells restored pexidartinib-induced cell-cycle arrest but not apoptosis. CONCLUSIONS: Mutations in CCND3, a gene not commonly mutated in AML, are a novel cause of clinical primary resistance to FLT3 inhibitors in AML and may have sensitivity to CDK4/6 inhibition.

A novel combination regimen of BET and FLT3 inhibition for FLT3-ITD acute myeloid leukemia.

Acute myeloid leukemia patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors improve overall survival, but their efficacy is limited and most patients who relapse will ultimately die of the disease. Even with potent FLT3 inhibition, the disease persists within the bone marrow microenvironment, mainly due to bone marrow stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as MYC and BCL2, but these drugs thus far have shown only limited single-agent clinical potential. We demonstrate here, using pre-clinical and clinical correlative studies, that the novel 4-azaindole derivative, PLX51107, has BET-inhibitory activity in vitro and in vivo. The combination of BET and FLT3 inhibition induces a synergistic antileukemic effect in a murine xenograft model of FLT3-ITD AML, and against primary FLT3-ITD AML cells co-cultured with bone marrow stroma. Using suppression of MYC as a surrogate for BET inhibition, we demonstrate BET inhibition in human patients. The short plasma half-life of PLX51107 results in intermittent target inhibition to enable tolerability while overcoming the protective effect of the microenvironment. Mechanistically, the synergistic cytotoxicity is associated with suppression of key survival genes such as MYC. These data provide the scientific rationale for a clinical trial of a BET plus FLT3 inhibitor for the treatment of relapsed/refractory FLT3-ITD AML. A clinical trial of PLX51107 as monotherapy in patients with different malignancies is underway and will be reported separately.

A phase 1/2 study of the oral FLT3 inhibitor pexidartinib in relapsed/refractory FLT3-ITD-mutant acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have activity in acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (ITD) mutations, but efficacy is limited by resistance-conferring kinase domain mutations. This phase 1/2 study evaluated the safety, tolerability, and efficacy of the oral FLT3 inhibitor PLX3397 (pexidartinib), which has activity against the FLT3 TKI-resistant F691L gatekeeper mutation in relapsed/refractory FLT3-ITD-mutant AML. Ninety patients were treated: 34 in dose escalation (part 1) and 56 in dose expansion (part 2). Doses of 800 to 5000 mg per day in divided doses were tested. No maximally tolerated dose was reached. Plasma inhibitory assay demonstrated that patients dosed with ≥3000 mg had sufficient levels of active drug in their trough plasma samples to achieve 95% inhibition of FLT3 phosphorylation in an FLT3-ITD AML cell line. Based on a plateau in drug exposure, the 3000-mg dose was chosen as the recommended phase 2 dose. The most frequently reported treatment-emergent adverse events were diarrhea (50%), fatigue (47%), and nausea (46%). Based on modified response criteria, the overall response rate to pexidartinib among all patients was 21%. Twenty-three percent of patients treated at ≥2000 mg responded. The overall composite complete response rate for the study was 11%. Six patients were successfully bridged to transplantation. Median overall survival (OS) of patients treated in dose expansion was 112 days (90% confidence interval [CI], 77-150 days), and median OS of responders with complete remission with or without recovery of blood counts was 265 days (90% CI, 170-422 days). This trial was registered at www.clinicaltrials.gov as #NCT01349049.

Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model.

Many risk genes for the development of Alzheimer's disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aß deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis.

Inhibition of TAMs improves the response to docetaxel in castration-resistant prostate cancer.

For men with castration-resistant prostate cancer (CRPC), androgen-deprivation therapy (ADT) often becomes ineffective requiring the addition of docetaxel, a proven effective chemotherapy option. Tumor-associated macrophages (TAMs) are known to provide protumorigenic influences that contribute to treatment failure. In this study, we examined the contribution of TAMs to docetaxel treatment. An increased infiltration of macrophages in CRPC tumors was observed after treatment with docetaxel. Prostate cancer cells treated with docetaxel released more macrophage colony-stimulating factor (M-CSF-1 or CSF-1), IL-10 and other factors, which can recruit and modulate circulating monocytes to promote their protumorigenic functions. Inhibition of CSF-1 receptor kinase signaling with a small molecule antagonist (PLX3397) in CRPC models significantly reduces the infiltration of TAMs and their influences. As such, the addition of PLX3397 to docetaxel treatment resulted in a more durable tumor growth suppression than docetaxel alone. This study reveals a rational strategy to abrogate the influences of TAMs and extend the treatment response to docetaxel in CRPC.

RAF inhibitor PLX8394 selectively disrupts BRAF dimers and RAS-independent BRAF-mutant-driven signaling.

Activating BRAF mutants and fusions signal as RAS-independent constitutively active dimers with the exception of BRAF V600 mutant alleles which can function as active monomers1. Current RAF inhibitors are monomer selective, they potently inhibit BRAF V600 monomers but their inhibition of RAF dimers is limited by induction of negative cooperativity when bound to one site in the dimer1-3. Moreover, acquired resistance to these drugs is usually due to molecular lesions that cause V600 mutants to dimerize4-8. We show here that PLX8394, a new RAF inhibitor9, inhibits ERK signaling by specifically disrupting BRAF-containing dimers, including BRAF homodimers and BRAF-CRAF heterodimers, but not CRAF homodimers or ARAF-containing dimers. Differences in the amino acid residues in the amino (N)-terminal portion of the kinase domain of RAF isoforms are responsible for this differential vulnerability. As a BRAF-specific dimer breaker, PLX8394 selectively inhibits ERK signaling in tumors driven by dimeric BRAF mutants, including BRAF fusions and splice variants as well as BRAF V600 monomers, but spares RAF function in normal cells in which CRAF homodimers can drive signaling. Our work suggests that drugs with these properties will be safe and useful for treating tumors driven by activating BRAF mutants or fusions.

PLX9486 shows anti-tumor efficacy in patient-derived, tyrosine kinase inhibitor-resistant KIT-mutant xenograft models of gastrointestinal stromal tumors.

The purpose of the present study was to investigate the in vitro and in vivo activity of PLX9486, a tyrosine kinase inhibitor (TKI) targeting both primary KIT exon 9 and 11 and secondary exon 17 and 18 mutations in gastrointestinal stromal tumors (GISTs). Imatinib, a potent inhibitor of mutated KIT, has revolutionized the clinical management of advanced, metastatic GIST. However, secondary resistance develops mainly through acquired mutations in KIT exons 13/14 or exons 17/18. Second-line sunitinib potently inhibits KIT exon 13/14 mutants but is ineffective against exon 17 mutations. In our study, PLX9486 demonstrated in vitro nanomolar potency in inhibiting the growth and KIT phosphorylation of engineered BaF3 cells transformed with KIT exon 17 mutations (p.D816V) and with the double KIT exon 11/17 mutations (p.V560G/D816V). The in vivo efficacy of PLX9486 was evaluated using two imatinib-resistant GIST patient-derived xenograft (PDX) models. In UZLX-GIST9 (KIT: p.P577del;W557LfsX5;D820G), PLX9486 100 mg/kg/day resulted in significant inhibition of proliferation. Pharmacodynamic analysis showed a pronounced reduction in mitogen-activated protein kinase (MAPK) activation and other downstream effects of the KIT signaling pathway but no significant effect on KIT Y703 and Y719 phosphorylation. Similarly, in MRL-GIST1 (KIT: p.W557_K558del;Y823D) PLX9486 treatment led to significant tumor regression and strong inhibition of MAPK activation. Interestingly, the inhibitory effect on MAPK activation was evident even after a single dose of PLX9486. In conclusion, PLX9486 showed anti-tumor efficacy in patient-derived imatinib-resistant GIST xenograft models, mainly through inhibition of KIT signaling. These preclinical efficacy data encourage further testing of PLX9486 in the clinical setting.

The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma.

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 of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.

BRD4 Profiling Identifies Critical Chronic Lymphocytic Leukemia Oncogenic Circuits and Reveals Sensitivity to PLX51107, a Novel Structurally Distinct BET Inhibitor.

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 overexpressed in CLL and is enriched proximal to genes upregulated or de novo expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a 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 preclinical 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 BRD4 profiling in other cancers.Significance: To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. Cancer Discov; 8(4); 458-77. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

Response and Resistance to Paradox-Breaking BRAF Inhibitor in Melanomas In Vivo and Ex Vivo.

FDA-approved BRAF inhibitors produce high response rates and improve overall survival in patients with BRAF V600E/K-mutant melanoma, but are linked to pathologies associated with paradoxical ERK1/2 activation in wild-type BRAF cells. To overcome this limitation, a next-generation paradox-breaking RAF inhibitor (PLX8394) has been designed. Here, we show that by using a quantitative reporter assay, PLX8394 rapidly suppressed ERK1/2 reporter activity and growth of mutant BRAF melanoma xenografts. Ex vivo treatment of xenografts and use of a patient-derived explant system (PDeX) revealed that PLX8394 suppressed ERK1/2 signaling and elicited apoptosis more effectively than the FDA-approved BRAF inhibitor, vemurafenib. Furthermore, PLX8394 was efficacious against vemurafenib-resistant BRAF splice variant-expressing tumors and reduced splice variant homodimerization. Importantly, PLX8394 did not induce paradoxical activation of ERK1/2 in wild-type BRAF cell lines or PDeX. Continued in vivo dosing of xenografts with PLX8394 led to the development of acquired resistance via ERK1/2 reactivation through heterogeneous mechanisms; however, resistant cells were found to have differential sensitivity to ERK1/2 inhibitor. These findings highlight the efficacy of a paradox-breaking selective BRAF inhibitor and the use of PDeX system to test the efficacy of therapeutic agents. Mol Cancer Ther; 17(1); 84-95. ©2017 AACR.

Anaphase-Promoting Complex Adaptor FZR1/CDH1 Blocks BRAF Signaling Both by Targeting BRAF for Proteolytic Degradation and by Disrupting BRAF Dimerization.

Blocking dimerization and stimulating protein degradation are two mechanisms known to inhibit BRAF activity. The study reported by Wan and colleagues identifies BRAF as a substrate of the APC/CFZR1-ubiqutin-proteasome system. The interaction between FZR1 and BRAF also induces a conformational change that disrupts BRAF dimerization. These findings identify a dynamic interplay between FZR1 and BRAF with strong implications for cell-fate determination and the tumor suppressor role of FZR1. Cancer Discov; 7(4); 356-8. ©2017 AACRSee related article by Wan et al., p. 424.

Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer.

Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ∼2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAFV600E-mutant allele, the spectrum of BRAF mutations in LA includes BRAFV600E (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAFG469A and BRAFG466V The presence of BRAFV600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAFV600E-mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAFV600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAFV600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.