Convergent MAPK pathway alterations mediate acquired resistance to FGFR inhibitors in FGFR2 fusion-positive cholangiocarcinoma.

BACKGROUND & AIMS: There is a knowledge gap in understanding mechanisms of resistance to fibroblast growth factor receptor (FGFR) inhibitors (FGFRi) and a need for novel therapeutic strategies to overcome it. We investigated mechanisms of acquired resistance to FGFRi in patients with FGFR2-fusion-positive cholangiocarcinoma (CCA). METHODS: A retrospective analysis of patients who received FGFRi therapy and underwent tumor and/or cell-free DNA analysis, before and after treatment, was performed. Longitudinal circulating tumor DNA samples from a cohort of patients in the phase I trial of futibatinib (NCT02052778) were assessed. FGFR2-BICC1 fusion cell lines were developed and secondary acquired resistance mutations in the mitogen-activated protein kinase (MAPK) pathway were introduced to assess their effect on sensitivity to FGFRi in vitro. RESULTS: On retrospective analysis of 17 patients with repeat sequencing following FGFRi treatment, new FGFR2 mutations were detected in 11 (64.7%) and new alterations in MAPK pathway genes in nine (52.9%) patients, with seven (41.2%) patients developing new alterations in both the FGFR2 and MAPK pathways. In serially collected plasma samples, a patient treated with an irreversible FGFRi tested positive for previously undetected BRAF V600E, NRAS Q61K, NRAS G12C, NRAS G13D and KRAS G12K mutations upon progression. Introduction of a FGFR2-BICC1 fusion into biliary tract cells in vitro sensitized the cells to FGFRi, while concomitant KRAS G12D or BRAF V600E conferred resistance. MEK inhibition was synergistic with FGFRi in vitro. In an in vivo animal model, the combination had antitumor activity in FGFR2 fusions but was not able to overcome KRAS-mediated FGFRi resistance. CONCLUSIONS: These findings suggest convergent genomic evolution in the MAPK pathway may be a potential mechanism of acquired resistance to FGFRi. CLINICAL TRIAL NUMBER: NCT02052778. IMPACT AND IMPLICATIONS: We evaluated tumors and plasma from patients who previously received inhibitors of fibroblast growth factor receptor (FGFR), an important receptor that plays a role in cancer cell growth, especially in tumors with abnormalities in this gene, such as FGFR fusions, where the FGFR gene is fused to another gene, leading to activation of cancer cell growth. We found that patients treated with FGFR inhibitors may develop mutations in other genes such as KRAS, and this can confer resistance to FGFR inhibitors. These findings have several implications for patients with FGFR2 fusion-positive tumors and provide mechanistic insight into emerging MAPK pathway alterations which may serve as a therapeutic vulnerability in the setting of acquired resistance to FGFRi.

MEKK2 regulates focal adhesion stability and motility in invasive breast cancer cells.

MEK Kinase 2 (MEKK2) is a serine/threonine kinase that functions as a MAPK kinase kinase (MAP3K) to regulate activation of Mitogen-activated Protein Kinases (MAPKs). We recently have demonstrated that ablation of MEKK2 expression in invasive breast tumor cells dramatically inhibits xenograft metastasis, but the mechanism by which MEKK2 influences metastasis-related tumor cell function is unknown. In this study, we investigate MEKK2 function and demonstrate that silencing MEKK2 expression in breast tumor cell significantly enhances cell spread area and focal adhesion stability while reducing cell migration. We show that cell attachment to the matrix proteins fibronectin or Matrigel induces MEKK2 activation and localization to focal adhesions. Further, we reveal that MEKK2 ablation enhances focal adhesion size and frequency, thereby linking MEKK2 function to focal adhesion stability. Finally, we show that MEKK2 knockdown inhibits fibronectin-induced Extracellular Signal-Regulated Kinase 5 (ERK5) signaling and Focal Adhesion Kinase (FAK) autophosphorylation. Taken together, our results strongly support a role for MEKK2 as a regulator of signaling that modulates breast tumor cell spread area and migration through control of focal adhesion stability.

MEKK2 regulates paxillin ubiquitylation and localization in MDA-MB 231 breast cancer cells.

The intracellular kinase MEKK2 (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase 2) is an upstream regulator of JNK (c-Jun N-terminal kinase), but additional functions for MEKK2 have not been well defined. Silencing MEKK2 expression in invasive breast tumour cells markedly inhibits xenograft metastasis, indicating that MEKK2 controls tumour cell function required for tumour progression. In our previous investigation of MEKK2 function, we discovered that tumour cell attachment to fibronectin recruits MEKK2 to focal adhesion complexes, and that MEKK2 knockdown is associated with stabilized focal adhesions and significant inhibition of tumour cell migration. In the present study we investigate MEKK2 function in focal adhesions and we report that MEKK2 physically associates with the LD1 motif of the focal adhesion protein paxillin. We reveal that MEKK2 induces paxillin ubiquitylation, and that this function requires both the paxillin LD1 motif and MEKK2 kinase activity. Finally, we demonstrate that MEKK2 promotes paxillin redistribution from focal adhesions into the cytoplasm, but does not promote paxillin degradation. Taken together, our results reveal a novel function for MEKK2 as a regulator of ubiquitylation-dependent paxillin redistribution in breast tumour cells.

Perlecan Domain-V Enhances Neurogenic Brain Repair After Stroke in Mice.

The extracellular matrix fragment perlecan domain V is neuroprotective and functionally restorative following experimental stroke. As neurogenesis is an important component of chronic post-stroke repair, and previous studies have implicated perlecan in developmental neurogenesis, we hypothesized that domain V could have a broad therapeutic window by enhancing neurogenesis after stroke. We demonstrated that domain V is chronically increased in the brains of human stroke patients, suggesting that it is present during post-stroke neurogenic periods. Furthermore, perlecan deficient mice had significantly less neuroblast precursor cells after experimental stroke. Seven-day delayed domain V administration enhanced neurogenesis and restored peri-infarct excitatory synaptic drive to neocortical layer 2/3 pyramidal neurons after experimental stroke. Domain V's effects were inhibited by blockade of α2ß1 integrin, suggesting the importance of α2ß1 integrin to neurogenesis and domain V neurogenic effects. Our results demonstrate that perlecan plays a previously unrecognized role in post-stroke neurogenesis and that delayed DV administration after experimental stroke enhances neurogenesis and improves recovery in an α2ß1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window.

Identification of Actionable Genomic Alterations Using Circulating Cell-Free DNA.

PURPOSE: Cell-free DNA (cfDNA) next-generation sequencing is a noninvasive approach for genomic testing. We report the frequency of identifying alterations and their clinical actionability in patients with advanced/metastatic cancer. PATIENTS AND METHODS: Prospectively consented patients had cfDNA testing performed. Alterations were assessed for therapeutic implications. RESULTS: We enrolled 575 patients with 37 tumor types. Of these patients, 438 (76.2%) had at least one alteration detected, and 205 (35.7%) had one or more alterations of high potential for clinical action. In diseases with 10 or more patients enrolled, 50% or more had at least one alteration deemed of high potential for clinical action. Trials were identified in 80% of patients (286 of 357) with any alteration and in 92% of patients (188 of 205) with one or more alterations of high potential for clinical action of whom 57.6% (118 of 205) had 6 or more months of follow-up available. Of these patients, 10% (12 of 118) had received genomically matched therapy through enrollment in clinical trials (n = 8), off-label drug use (n = 3), or standard of care (n = 1). Although 88.6% of all patients had a performance status of 0 or 1 upon enrollment, the primary reason for not acting on alterations was poor performance status at next treatment change (28.1%; 27 of 96). CONCLUSION: cfDNA testing represents a readily accessible method for genomic testing and allows for detection of genomic alterations in most patients with advanced disease. Utility may be higher in patients interested in investigational therapeutics with adequate performance status. Additional study is needed to determine whether utility is enhanced by testing earlier in the treatment course.

CXCR7 Reactivates ERK Signaling to Promote Resistance to EGFR Kinase Inhibitors in NSCLC.

Although EGFR mutant-selective tyrosine kinase inhibitors (TKI) are clinically effective, acquired resistance can occur by reactivating ERK. We show using in vitro models of acquired EGFR TKI resistance with a mesenchymal phenotype that CXCR7, an atypical G protein-coupled receptor, activates the MAPK-ERK pathway via ß-arrestin. Depletion of CXCR7 inhibited the MAPK pathway, significantly attenuated EGFR TKI resistance, and resulted in mesenchymal-to-epithelial transition. CXCR7 overexpression was essential in reactivation of ERK1/2 for the generation of EGFR TKI-resistant persister cells. Many patients with non-small cell lung cancer (NSCLC) harboring an EGFR kinase domain mutation, who progressed on EGFR inhibitors, demonstrated increased CXCR7 expression. These data suggest that CXCR7 inhibition could considerably delay and prevent the emergence of acquired EGFR TKI resistance in EGFR-mutant NSCLC. SIGNIFICANCE: Increased expression of the chemokine receptor CXCR7 constitutes a mechanism of resistance to EGFR TKI in patients with non-small cell lung cancer through reactivation of ERK signaling.

Interaction with the Paxillin LD1 Motif Relieves MEKK2 Auto-inhibition.

The cell signaling molecule MEK kinase 2 (MEKK2) is a key upstream regulator of MAPK activity that regulates numerous cellular functions, but the mechanisms that control MEKK2 activity are not well understood. Recently, we reported that MEKK2 both binds and promotes ubiquitylation of the scaffold protein paxillin, and thereby modulates the composition of adhesion complexes. In this study, we have extended our examination of this interaction and report that recombinant paxillin is sufficient to induce MEKK2 auto-phosphorylation. Furthermore, we utilize siRNA-mediated paxillin expression knockdown to reveal that MEKK2 activity is reduced in paxillin-deficient cells. Finally, we show that the paxillin leucine-rich motif 1 (LD1) is sufficient to bind to the MEKK2 amino terminal region and activate MEKK2. Taken together, our results show for the first time that paxillin association promotes MEKK2 activation and reveal the existence of a novel bi-directional regulatory relationship between MEKK2 and paxillin.

Neuronal restoration following ischemic stroke: influences, barriers, and therapeutic potential.

Neurogenesis, the birth of new neurons, occurs throughout life in the subventricular zone and produces immature neurons that migrate tangentially through the rostral migratory stream to the olfactory bulb. This migration is tightly regulated by both structural and chemical influences. Interestingly, brain insults such as ischemic stroke increase neurogenesis and redirect neuroblast migration to the injury site. This injury-redirected neurogenesis and migration is coupled with angiogenic vasculature and is influenced by many of the factors that positively and negatively affect migration under developmental or normal adult conditions. Additionally, cytokines and chemokines such as stromal cell-derived factor-1 strongly influence neuronal migration poststroke. However, neuronal repopulation or brain regeneration is extremely limited. This limitation may potentially be due to the hostile poststroke microenvironment including the formation of the physical and chemical barriers of glial scar. Furthermore, interspecies differences in poststroke neurogenesis between rodents and humans complicate the translation of experimental results to humans. Despite these challenges, many drugs and other potential therapies have recently been evaluated for potential neurogenic properties poststroke. Improved understanding of poststroke neurorepair may lead to new and more effective neurorestorative therapies.

Successfully Climbing the "STAIRs": Surmounting Failed Translation of Experimental Ischemic Stroke Treatments.

The Stroke Therapy Academic Industry Roundtable (STAIR) provided initial (in 1999) and updated (in 2009) recommendations with the goal of improving preclinical stroke therapy assessment and to increase the translational potential of experimental stroke treatments. It is important for preclinical stroke researchers to frequently consider and revisit these concepts, especially since promising experimental stroke treatments continue to fail in human clinical trials. Therefore, this paper will focus on considerations for several key aspects of preclinical stroke studies including the selection and execution of the animal stroke model, drug/experimental treatment administration, and outcome measures to improve experimental validity and translation potential. Specific points of interest discussed include the incorporation of human comorbid conditions and drugs, the benefits of defining a proposed mechanism of action, replication of results using multiple methods, using clinically relevant routes of administration and treatment time windows, and performing and reporting good experimental methods to reduce bias such as, as suggested by the updated STAIR recommendations, sample size calculations, randomization, allocation concealment, blinding, and appropriate inclusion/exclusion criteria. It is our hope that reviewing and revisiting these considerations will benefit researchers in their investigations of stroke therapies and increase the likelihood of translational success in the battle against stroke.

Perlecan and the blood-brain barrier: beneficial proteolysis?

The cerebral microvasculature is important for maintaining brain homeostasis. This is achieved via the blood-brain barrier (BBB), composed of endothelial cells with specialized tight junctions, astrocytes, and a basement membrane (BM). Prominent components of the BM extracellular matrix (ECM) include fibronectin, laminin, collagen IV, and perlecan, all of which regulate cellular processes via signal transduction through various cell membrane bound ECM receptors. Expression and proteolysis of these ECM components can be rapidly altered during pathological states of the central nervous system. In particular, proteolysis of perlecan, a heparan sulfate proteoglycan, occurs within hours following ischemia induced by experimental stroke. Proteolysis of ECM components following stroke results in the degradation of the BM and further disruption of the BBB. While it is clear that such proteolysis has negative consequences for the BBB, we propose that it also may lead to generation of ECM protein fragments, including the C-terminal domain V (DV) of perlecan, that potentially have a positive influence on other aspects of CNS health. Indeed, perlecan DV has been shown to be persistently generated after stroke and beneficial as a neuroprotective molecule and promoter of post-stroke brain repair. This mini-review will discuss beneficial roles of perlecan protein fragment generation within the brain during stroke.

Perlecan domain V is upregulated in human brain arteriovenous malformation and could mediate the vascular endothelial growth factor effect in lesional tissue.

Brain arteriovenous malformation (BAVM), a rare but important cause of intracranial hemorrhage, has increased angiogenesis and inflammation as key components of the nidus of abnormal vessels and stroma that form the resected surgical specimen. Accordingly, both vascular endothelial growth factor (VEGF) and transforming growth factor-ß have been implicated in the pathology of BAVM for their proangiogenic and vascular-regulating roles. The C-terminal fragment of the extracellular matrix component perlecan (domain V, DV) has been shown to be increased and through the α5ß1 integrin, to increase VEGF levels in and around areas of cerebral ischemic injury, another proangiogenic condition. We aimed to determine whether the concentrations of DV, DV's proangiogenic receptor α5ß1 integrin, or DV's antiangiogenic receptor α2ß1 integrin are elevated in human BAVM tissue. DV levels were increased in BAVM compared with control brain tissue from epileptic resection, as was α5ß1 integrin. In addition, α5ß1 integrin was preferentially increased and localized to endothelial cells compared with α2ß1 integrin. VEGF and transforming growth factor-ß levels were also increased in BAVM compared with control tissue. Furthermore, increases in all components were strongly correlated. Excessive generation of proangiogenic DV in BAVM suggests that DV may participate in its pathology and may represent a future therapeutic target.