Author Correction: PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

In this Letter, two relevant references were omitted; see the accompanying Amendment for details. The original Letter has not been corrected.

PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

Biomechanical effects of muscle loading on early healing of femoral stem fractures: a combined musculoskeletal dynamics and finite element approach.

Femoral stem fractures (FST) are often accompanied by muscle injuries, however, what muscle injuries affect fracture healing and to what extent is unknown. The purpose of this study was to analyze the extent to which different muscles affect FST healing through a combined musculoskeletal dynamics and finite element approach. Modeling the lower extremity musculoskeletal system for 12 different muscle comprehensives. Muscle and joint reaction forces on the femur were calculated and these data were used as boundary conditions input to the FSTs model to predict the degree of muscle influence on fracture healing. Finally, we will investigate the extent to which muscle influences FST healing during knee flexion. Muscle and joint forces are highly dependent on joint motion and have a significant biomechanical influence on interfragmentary strain (IFS) healing. The psoas major (PM), gastrocnemius lateralis (GL) and gastrocnemius medialis (GM) muscles play a major role in standing, with GM > PM > GL, whereas the gluteus medius posterior (GMP), vastus intermedius (VI), vastus medialis (VM), vastus lateralis superior (VLS), and adductor magnus distalis (AMD) muscles play a major role in knee flexion, with VLS > VM > VI > AMD > GMP. Mechanical stimulus-controlled healing can be facilitated when the knee joint is flexed less than 20°. Different muscles exert varying degrees of influence on the healing of fractures. Therefore, comprehending the impact of particular muscles on fracture site tissue FST healing can aid orthopedic surgeons in formulating improved surgical and rehabilitation strategies.

Influence of knee flexion on early femoral fracture healing: A combined analysis of musculoskeletal dynamics and finite elements.

BACKGROUND AND OBJECTIVES: Knee flexion causes a certain amount of misalignment and relative movement of the fractured ends of the femur fracture, and if the flexion angle is too large it will affect the stability of the fracture and the healing process, making it challenging to design a safe range of flexion. However, due to a lack of basic understanding of the effect of knee flexion on the mechanical environment at the fracture site, clinicians are often unable to provide an objective and safe range of motion in flexion based on subjective experience. The aim of this study was to evaluate the effect of knee flexion on plate and fracture healing using finite element analysis (FEA). METHODS: A human musculoskeletal model was constructed based on CT scan data, and the mechanical properties of the fracture site were changed by adjusting the knee flexion angle. The joint forces, muscle forces and moments acting on the femur were obtained by inverse dynamics analysis, and the biomechanical properties of the fracture-plate system were analyzed using finite elements. A finite element model of the fracture-plate system without muscle loading was also constructed. The effect of knee flexion on the safety of plate fixation and fracture healing was evaluated in terms of the biomechanical properties of the plate and the interfragmentary motion of the fracture. RESULTS: As the knee flexion angle increases, the von Mises stress of the locked compression plate (LCP) first increases, then decreases, then increases again. The deformation from compression bending to tension twisting occurs simultaneously. At 30° of flexion, shear interfragmentary motion (SIM) was dominant and inhibited fracture healing; at more than 45° of flexion, the plate was twisted and deformed to the lateral side of the body, and the fracture site underwent greater misalignment and relative motion, with destructive effects on bone scabs and healing tissues. If muscle loading is not taken into account, the plate will undergo predominantly bending deformation and will overestimate the interfragmentary strain in the far and near cortex. CONCLUSIONS: Knee flexion causes the plate to deform from compression bending to extension and torsion, which has an important impact on the safety and healing process of the fracture, and this study provides a biomechanical basis to guide the clinician in the post-operative rehabilitation of femoral fractures in the clinical setting.

Procedures between training and reactivation influence the destabilization of instrumental sucrose memory.

Memory destabilization and reconsolidation is hypothesized to be a fundamental mnemonic process that can underpin memory updating. Instrumental memories have been shown recently to be destabilized following a reactivation session that involves a change in instrumental reward contingency. However, the acquisition and performance of an instrumental response occurs in the presence of the learning of other reward-related memories. This may influence the ability of a given reactivation session to destabilize the previously learned instrumental memory. Here we present a series of experiments in male rats involving an instrumental memory trained on an FR1 schedule over 10 days, and then reactivated in a session that imposed a VR5 schedule of reinforcement. When MK-801 was injected prior to the VR5 reactivation session, it reliably impaired subsequent instrumental performance at test only when the reactivation session occurred 48 h, and not 24 h, after the end of training. The interposition between the end of training and the reactivation session of a context extinction session, an additional VR5 reactivation session, or indeed the simple experience of being handled and injected with vehicle, resulted in MK-801 no longer having an amnestic effect on test performance. While we do not have a clear account for the process and mechanism underpinning this apparent selectivity of the effect of the VR5 session to destabilize the instrumental memory, it does additionally highlight the need for greater understanding of the conditions that facilitate reactivation-induced memory destabilization.

NUMB as a Therapeutic Target for Melanoma.

The upregulation of the adaptor protein NUMB triggers melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Although NUMB acts as a tumor suppressor in various human cancer types, little is known about its role in melanoma. In this study, we investigated the role of NUMB in melanoma progression and its regulatory mechanism. Analysis of The Cancer Genome Atlas melanoma datasets revealed that high NUMB expression in melanoma tissues correlates with improved patient survival. Moreover, NUMB expression is downregulated in metastatic melanoma cells. NUMB knockdown significantly increased the invasion potential of melanoma cells in a three-dimensional collagen matrix in vitro and in the lungs of a mouse model in vivo; it also significantly upregulated the expression of the NOTCH target gene CCNE. Previous studies suggested that Wnt signaling increases NUMB expression. By mimicking Wnt stimulation through glycogen synthase kinase-3 inhibition, we increased NUMB expression in melanoma cells. Furthermore, a glycogen synthase kinase-3 inhibitor reduced the invasion of melanoma cells in a NUMB-dependent manner. Together, our results suggest that NUMB suppresses invasion and metastasis in melanoma, potentially through its regulation of the NOTCH‒CCNE axis and that the inhibitors that upregulate NUMB can exert therapeutic effects in melanoma.

Neural Crest-Like Stem Cell Transcriptome Analysis Identifies LPAR1 in Melanoma Progression and Therapy Resistance.

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell-like pathways hijacked by tumor cells. SIGNIFICANCE: This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.

Enhancing KDM5A and TLR activity improves the response to immune checkpoint blockade.

Immune checkpoint blockade (ICB) therapies are now established as first-line treatments for multiple cancers, but many patients do not derive long-term benefit from ICB. Here, we report that increased amounts of histone 3 lysine 4 demethylase KDM5A in tumors markedly improved response to the treatment with the programmed cell death protein 1 (PD-1) antibody in mouse cancer models. In a screen for molecules that increased KDM5A abundance, we identified one (D18) that increased the efficacy of various ICB agents in three murine cancer models when used as a combination therapy. D18 potentiated ICB efficacy through two orthogonal mechanisms: (i) increasing KDM5A abundance, which suppressed expression of the gene PTEN (encoding phosphatase and tensin homolog) and increased programmed cell death ligand 1 abundance through a pathway involving PI3K-AKT-S6K1, and (ii) activating Toll-like receptors 7 and 8 (TLR7/8) signaling pathways. Combination treatment increased T cell activation and expansion, CD103+ tumor-infiltrating dendritic cells, and tumor-associated M1 macrophages, ultimately enhancing the overall recruitment of activated CD8+ T cells to tumors. In patients with melanoma, a high KDM5A gene signature correlated with KDM5A expression and could potentially serve as a marker of response to anti-PD-1 immunotherapy. Furthermore, our results indicated that bifunctional agents that enhance both KDM5A and TLR activity warrant investigation as combination therapies with ICB agents.

A Fatty Acid Oxidation-dependent Metabolic Shift Regulates the Adaptation of BRAF-mutated Melanoma to MAPK Inhibitors.

PURPOSE: Treatment of BRAFV600E -mutant melanomas with MAPK inhibitors (MAPKi) results in significant tumor regression, but acquired resistance is pervasive. To understand nonmutational mechanisms underlying the adaptation to MAPKi and to identify novel vulnerabilities of melanomas treated with MAPKi, we focused on the initial response phase during treatment with MAPKi. EXPERIMENTAL DESIGN: By screening proteins expressed on the cell surface of melanoma cells, we identified the fatty acid transporter CD36 as the most consistently upregulated protein upon short-term treatment with MAPKi. We further investigated the effects of MAPKi on fatty acid metabolism using in vitro and in vivo models and analyzing patients' pre- and on-treatment tumor specimens. RESULTS: Melanoma cells treated with MAPKi displayed increased levels of CD36 and of PPARα-mediated and carnitine palmitoyltransferase 1A (CPT1A)-dependent fatty acid oxidation (FAO). While CD36 is a useful marker of melanoma cells during adaptation and drug-tolerant phases, the upregulation of CD36 is not functionally involved in FAO changes that characterize MAPKi-treated cells. Increased FAO is required for BRAFV600E -mutant melanoma cells to survive under the MAPKi-induced metabolic stress prior to acquiring drug resistance. The upfront and concomitant inhibition of FAO, glycolysis, and MAPK synergistically inhibits tumor cell growth in vitro and in vivo. CONCLUSIONS: Thus, we identified a clinically relevant therapeutic approach that has the potential to improve initial responses and to delay acquired drug resistance of BRAFV600E -mutant melanoma.

Co-targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor-resistant melanoma.

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS-mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS-mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS-mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co-targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment-resistant tumor types.

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.

Induction of Telomere Dysfunction Prolongs Disease Control of Therapy-Resistant Melanoma.

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients.Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2'-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG.Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL.Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771-84. ©2018 AACR See related commentary by Teh and Aplin, p. 4629.