RAS signaling and therapeutic resistance in melanoma

Increasing appreciation of the complexity of RAS signaling in cancer has led to a renewed wave of RAS research. I have focused on two key areas: the role of wild-type RAS isoforms in RAS-mutant cancers, and mechanisms of resistance to molecularly targeted therapies directed against RAS effector pathways. Melanoma, the most aggressive form of skin cancer, presents an excellent model to study RAS signaling; ~90% of melanomas have a driver mutation in NRAS (26%) or BRAF (63%), thus hyper-activating the canonical RAS-RAF-MEK-ERK effector signaling pathway. An army of small molecule inhibitors has emerged to target this pathway, and several are FDA-approved for melanoma treatment. However, all targeted therapies face the challenge of resistance. Most validated mechanisms of resistance to these inhibitors involve either reactivation of ERK signaling or other bypass routes that result in cancer cell survival. In my investigation of resistance mechanisms in BRAF-mutant melanoma. I found that CK2α was sufficient to drive resistance to inhibitors of BRAF (BRAFi) and of MEK (MEKi). CK2α facilitated rebound of ERK phosphorylation in the presence of BRAFi, and maintained ERK phosphorylation upon treatment with MEKi. Surprisingly, by using a kinase-inactive mutant of CK2α, I showed that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold KSR1 suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. These findings support a new mechanism whereby a kinase-independent scaffolding function of CK2α promotes resistance to RAF- and MEK-targeted therapies. Another pressing issue is understanding the biological activities of wild-type RAS isoforms in RAS-mutant tumors. Most studies have investigated KRAS-mutant cancers, but little is known about NRAS-mutant cancers. NRAS-mutant melanomas comprise the second largest subgroup of melanoma patients, and no targeted therapy is approved for these patients. Exploring the roles of wild-type RAS isoforms in NRAS-mutant melanoma cells, I found that WT KRAS is essential for their proliferation and survival. Interestingly, depletion of KRAS resulted in a unique cellular morphology and in signaling outcomes distinct from those due to silencing of NRAS or HRAS. Moreover, KRAS knockdown stabilized p53 protein, which was accompanied by an increase in p53 target genes. Intriguingly, by using reverse phase protein array analysis, I found that KRAS knockdown severely impaired phosphorylation of ribosomal protein S6, dependent on S6K1 (p70 S6K) activity, but independent of Akt-mTOR, or ERK-p90RSK activity. These results may shed light on the potential efficacy of pan-RAS inhibition in NRAS-mutant cancers. Together, my findings uncover a novel kinase-independent scaffolding function of CK2α in promoting resistance to inhibitors of BRAF and MEK in BRAF-mutant melanoma; highlight the critical importance of WT KRAS in NRAS-mutant melanoma; and describe the unique role of WT KRAS in maintaining phosphorylation of ribosomal protein S6. How CK2α engages in scaffolding to maintain ERK signaling, and how KRAS circumvents Akt-mTOR to regulate ribosomal protein S6, are issues to be addressed in future studies of RAS signaling.Doctor of Philosoph

The role of wild type RAS isoforms in cancer

Mutationally activated RAS proteins are critical oncogenic drivers in nearly 30% of all human cancers. As with mutant RAS, the role of wild type RAS proteins in oncogenesis, tumour maintenance and metastasis is context-dependent. Complexity is introduced by the existence of multiple RAS genes (HRAS, KRAS, NRAS) and protein "isoforms" (KRAS4A, KRAS4B), by the ever more complicated network of RAS signaling, and by the increasing identification of numerous genetic aberrations in cancers that do and do not harbour mutant RAS. Numerous mouse model carcinogenesis studies and examination of patient tumours reveal that, in RAS-mutant cancers, wild type RAS proteins are likely to serve as tumour suppressors when the mutant RAS is of the same isoform. This evidence is particularly robust in KRAS mutant cancers, which often display suppression or loss of wild type KRAS, but is not as strong for NRAS. In contrast, although not yet fully elucidated, the preponderance of evidence indicates that wild type RAS proteins play a tumour promoting role when the mutant RAS is of a different isoform. In non-RAS mutant cancers, wild type RAS is recognized as a mediator of oncogenic signaling due to chronic activation of upstream receptor tyrosine kinases that feed through RAS. Additionally, in the absence of mutant RAS, activation of wild type RAS may drive cancer upon the loss of negative RAS regulators such as NF1 GAP or SPRY proteins. Here we explore the current state of knowledge with respect to the roles of wild type RAS proteins in human cancers

Advances in Treatment of Nocturnal Enuresis in Children

Nocturnal enuresis is a condition with complex etiology affecting plenty of children and families. Even though multifarious clinical trials and studies have been designed and completed, some inconclusive results on nocturnal enuresis confuse clinicians. This article aims to provide useful information for clinicians by summarizing the existing evidence on nocturnal enuresis and discussing the effectiveness and safety of different treatments. Nocturnal enuresis mainly results from the disorders related to central nervous system, which may cause nocturnal polyuria, nighttime bladder capacity decline, arousal disorder, and various accompanying diseases. We discussed the efficacy and safety of different treatments for monosymptomatic nocturnal enuresis, including standard therapies, simple behavioral interventions, complex behavioral interventions, alarm therapy, desmopressin and other drugs, biofeedback therapy, electrical stimulation, acupuncture, Chinese herbal medicine, massage, and so on. Alarm is still the most effective single therapy with lower relapse rate. Desmopressin has efficacy mainly in children with nocturnal polyuria. Children with detrusor overactivity or decreasing functional bladder capacity can choose anticholinergics. Additionally, tricyclic drugs, biofeedback therapy, electrical stimulation, acupuncture, massage, and so on are therapeutic options for children with nocturnal enuresis

Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma

The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies

Surface-guided computing to analyze subcellular morphology and membrane-associated signals in 3D

Signal transduction and cell function are governed by the spatiotemporal organization of membrane-associated molecules. Despite significant advances in visualizing molecular distributions by 3D light microscopy, cell biologists still have limited quantitative understanding of the processes implicated in the regulation of molecular signals at the whole cell scale. In particular, complex and transient cell surface morphologies challenge the complete sampling of cell geometry, membrane-associated molecular concentration and activity and the computing of meaningful parameters such as the cofluctuation between morphology and signals. Here, we introduce u-Unwrap3D, a framework to remap arbitrarily complex 3D cell surfaces and membrane-associated signals into equivalent lower dimensional representations. The mappings are bidirectional, allowing the application of image processing operations in the data representation best suited for the task and to subsequently present the results in any of the other representations, including the original 3D cell surface. Leveraging this surface-guided computing paradigm, we track segmented surface motifs in 2D to quantify the recruitment of Septin polymers by blebbing events; we quantify actin enrichment in peripheral ruffles; and we measure the speed of ruffle movement along topographically complex cell surfaces. Thus, u-Unwrap3D provides access to spatiotemporal analyses of cell biological parameters on unconstrained 3D surface geometries and signals.Comment: 49 pages, 10 figure

Strong Interplay between Stripe Spin Fluctuations, Nematicity and Superconductivity in FeSe

Elucidating the microscopic origin of nematic order in iron-based superconducting materials is important because the interactions that drive nematic order may also mediate the Cooper pairing. Nematic order breaks fourfold rotational symmetry in the iron plane, which is believed to be driven by either orbital or spin degrees of freedom. However, as the nematic phase often develops at a temperature just above or coincides with a stripe magnetic phase transition, experimentally determining the dominant driving force of nematic order is difficult. Here, we use neutron scattering to study structurally the simplest iron-based superconductor FeSe, which displays a nematic (orthorhombic) phase transition at $T_s=90$ K, but does not order antiferromagnetically. Our data reveal substantial stripe spin fluctuations, which are coupled with orthorhombicity and are enhanced abruptly on cooling to below $T_s$. Moreover, a sharp spin resonance develops in the superconducting state, whose energy (~4 meV) is consistent with an electron boson coupling mode revealed by scanning tunneling spectroscopy, thereby suggesting a spin fluctuation-mediated sign-changing pairing symmetry. By normalizing the dynamic susceptibility into absolute units, we show that the magnetic spectral weight in FeSe is comparable to that of the iron arsenides. Our findings support recent theoretical proposals that both nematicity and superconductivity are driven by spin fluctuations.Comment: 19 pages, 8 figure

INO80 governs superenhancer-mediated oncogenic transcription and tumor growth in melanoma

Superenhancers (SEs) are large genomic regions with a high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared with normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, tumorigenesis, and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies >90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are selectively expressed in melanomas compared with melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function and suggest a novel strategy for disrupting SEs in cancer treatment

The THO Complex Regulates Pluripotency Gene mRNA Export and Controls Embryonic Stem Cell Self-Renewal and Somatic Cell Reprogramming

Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, post-transcriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5, and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, as its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of post-transcriptional regulation in stem cell fate specification

INO80 Facilitates Pluripotency Gene Activation in Embryonic Stem Cell Self-Renewal, Reprogramming, and Blastocyst Development

The master transcription factors play integral roles in the pluripotency transcription circuitry of embryonic stem cells (ESCs). How they selectively activate expression of the pluripotency network while simultaneously repressing genes involved in differentiation is not fully understood. Here we define a requirement for the INO80 complex, a SWI/SNF family chromatin remodeler, in ESC self-renewal, somatic cell reprogramming, and blastocyst development. We show that Ino80, the chromatin remodeling ATPase, co-occupies pluripotency gene promoters with the master transcription factors, and its occupancy is dependent on Oct4 and Wdr5. At the pluripotency genes, Ino80 maintains open chromatin architecture and licenses recruitment of Mediator and RNA Polymerase II for gene activation. Our data reveal an essential role for INO80 in the expression of the pluripotency network, and illustrate the coordination among chromatin remodeler, transcription factor, and histone modifying enzyme in the regulation of the pluripotent state