KSR1 regulates small-cell lung carcinoma tumor initiation and cisplatin resistance.

Small-cell lung cancer (SCLC) is designated a recalcitrant cancer due to its five-year relative survival rate of less than 7%. First line SCLC treatment has changed modestly in the last 40 years. The NeuroD1 subtype of SCLC (SCLC-N) commonly harbors MYC amplifications and other hallmarks of aggressive behavior. Finding novel therapeutic options that effectively eliminate residual disease observed after initial response to therapy is essential to improving SCLC patient outcome. Here we show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK signaling cascade is critical for clonogenicity and tumor initiation in vitro and in vivo in the highly aggressive, metastatic and therapy resistant NeuroD1 subtype of SCLC. Tumor-initiating cells (TICs) are reported as the sanctuary population within the bulk tumor responsible for therapeutic resistance and relapse. Previous studies concluded ERK activation was inhibitory to growth and tumor development. We show that signaling through KSR1 is conserved in SCLC-N and that it regulates tumor initiation through interaction with ERK. We further show that KSR1 mediates cisplatin resistance in SCLC-N cells. While 50% of control SCLC-N cells show resistance after 6 weeks of exposure to cisplatin, CRISPR/Cas9-mediated KSR1 knockout prevents resistance in >90% of SCLC-N cells. KSR1 KO also significantly enhances the ability of cisplatin to decrease SCLC-N TICs, indicating that targeting KSR1 might be selectively toxic to cells responsible for therapeutic resistance and tumor initiation. Thus, KSR1 function in SCLC-N serves as a novel model for understanding the role of KSR1-dependent signaling in normal and malignant tissues. These findings shed light on a key distinct protein responsible for regulation in SCLC-N tumors, and a potential subtype specific therapeutic target.

SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status.

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor-initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 (Son of Sevenless 1) or KSR1 (Kinase Suppressor of RAS 1) both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid-initiating cells in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA comutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wild-type cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS-mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying comutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations.

PGC-1ß and ERRα Promote Glutamine Metabolism and Colorectal Cancer Survival via Transcriptional Upregulation of PCK2.

BACKGROUND: Previous studies have shown that Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Beta (PGC-1ß) and Estrogen-Related Receptor Alpha (ERRα) are over-expressed in colorectal cancer and promote tumor survival. METHODS: In this study, we use immunoprecipitation of epitope tagged endogenous PGC-1ß and inducible PGC-1ß mutants to show that amino acid motif LRELL on PGC-1ß is responsible for the physical interaction with ERRα and promotes ERRα mRNA and protein expression. We use RNAsequencing to determine the genes regulated by both PGC-1ß & ERRα and find that mitochondrial Phosphoenolpyruvate Carboxykinase 2 (PCK2) is the gene that decreased most significantly after depletion of both genes. RESULTS: Depletion of PCK2 in colorectal cancer cells was sufficient to reduce anchorage-independent growth and inhibit glutamine utilization by the TCA cycle. Lastly, shRNA-mediated depletion of ERRα decreased anchorage-independent growth and glutamine metabolism, which could not be rescued by plasmid derived expression of PCK2. DISCUSSION: These findings suggest that transcriptional control of PCK2 is one mechanism used by PGC-1ß and ERRα to promote glutamine metabolism and colorectal cancer cell survival.

Association of Serum Iron Studies in COVID Associated Mucormycosis with Stage of the Disease.

Mucormycosis (Zygomycosis) is a rare and lethal invasive fungal infection, often acute and extremely severe caused by opportunist and ubiquitous fungi belonging to the class Phygomycetes, subclass Zygomycetes, order Mucorales, family Mucoraceae. India has reported surge in cases of post COVID 19 Mucormycosis over the past few months due to the increasing frequency of risk factors like corticosteroid therapy, uncontrolled diabetes, DKA, neutropenia and iron overload. Patients with a history of COVID-19 infection are at increased risk of developing fungal infections like Mucormycosis. The emergence of COVID-19 associated mucormycosis (CAM) across several nations, particularly India, warrants a detailed study to identify potential contributing factors. MATERIAL: This cross sectional study conducted at Bowring and Lady Curzon Hospital, Bangalore, involving 75 subjects diagnosed with CAM either clinically, radiological or microbiologically. The objective was to study the clinical profile of patients with COVID associated Mucormycosis and to correlate the levels of Serum ferritin and iron profile with severity and extent of disease in COVID associated Mucormycosis patients Data was collected on demographic details, co morbidities, vaccination status, history of treatment with remedesvir, oxygen therapy or steroid use, complications of past COVID 19 infection and stage of current Mucormycosis infection. Clinical outcome of the patients measured based on Iron profile, length of hospital stay, need for ICU admission, presence of diabetic ketoacidosis and mortality. The blood investigations which included were CBC with differential leukocyte count, qCRP, FBS, PPBS, HbA1c serum iron studies and serum ferritin. OBSERVATION: The mean age of the subjects was 48.19 with 52 males, 23 females. Among 75 patients with CAM, 90.7% were unvaccinated against COVID-19, 62.7% had oxygen usage and steroid therapy, 44% had use of remedesvir. Most common co morbidity was diabetes mellitus 60% with 20% of patients having DKA. Rhino orbital-cerebral mucormycosis(Stage 4- 44.6%) was the most common clinical presentation. The mean serum iron (50.37) and TIBC (255.37) were significantly higher in Stage 4 CAM cases compared with less invasive stage 2 CAM cases. Patients with Stage 4 CAM had elevated levels of inflammatory markers LDH (292) DDimer (457) CRP(74.64). Case fatality rates of CAM was 12%. CONCLUSION: The results of this study revealed significant correlation between the clinical severity of CAM and higher mortality, increased serum iron levels and inflammatory markers in this population of patients. Therefore, patients with elevated levels of available serum iron are uniquely susceptible to mucormycosis infection, suggesting dysregulated iron metabolism in its pathogenesis.

KSR1- and ERK-dependent translational regulation of the epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here, we show that KSR1 and ERK promote EMT-like phenotype through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT-like phenotype, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E- to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT-like phenotype via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

The majority of cancer deaths result from tumor cells spreading to other parts of the body via a process known as metastasis. 90% of all cancers originate in epithelial cells that line the inner and outer surface of organs in our bodies. Epithelial cells, however, are typically stationary and must undergo various chemical and physical changes to transform in to migratory cells that can invade other tissues. This transformation process alters the amount of protein cells use to interact with one another. For example, epithelial cells from the colon produce less of a protein called E-cadherin as they transition into migrating cancer cells and make another protein called N-cadherin instead. A protein called KSR1 is a key component of a signaling pathway that is responsible for generating the proteins colon cancer cells need to survive. But it is unknown which proteins KSR1 helps synthesize and whether it plays a role in the metastasis of colon cancer cells. To investigate this, Rao et al. studied the proteins generated by cancerous colon cells cultured in the laboratory, in the presence and absence of KSR1. The experiment showed that KSR1 increases the levels of a protein called EPSTI1, which colon cancer cells need to transform into migratory cells. Depleting KSR1 caused cancer cells to generate less EPSTI1 and to share more features with healthy cells, such as higher levels of E-cadherin on their surface and reduced mobility. Adding EPSTI1 to the cancer cells that lacked KSR1 restored the traits associated with metastasis, such as high levels of N-cadherin, and allowed the cells to move more easily. These findings suggest that KSR1 and EPSTI1 could be new drug targets for reducing, or potentially reversing, the invasive behavior of colon cancer cells. However, further investigation is needed to reveal how EPSTI1 is generated and how this protein helps colon cancer cells move and invade other tissues.

A Gene Expression High-Throughput Screen (GE-HTS) for Coordinated Detection of Functionally Similar Effectors in Cancer.

Genome-wide, loss-of-function screening can be used to identify novel vulnerabilities upon which specific tumor cells depend for survival. Functional Signature Ontology (FUSION) is a gene expression-based high-throughput screening (GE-HTS) method that allows researchers to identify functionally similar proteins, small molecules, and microRNA mimics, revealing novel therapeutic targets. FUSION uses cell-based high-throughput screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA libraries to identify putative protein targets and mechanisms of action (MoA) for several previously undescribed natural products. We have used FUSION to screen for functional analogues to Kinase suppressor of Ras 1 (KSR1), a scaffold protein downstream of Ras in the Raf-MEK-ERK kinase cascade, and biologically validated several proteins with functional similarity to KSR1. FUSION incorporates bioinformatics analysis that may offer higher resolution of the endpoint readout than other screens which utilize Boolean outputs regarding a single pathway activation (i.e., synthetic lethal and cell proliferation). Challenges associated with FUSION and other high-content genome-wide screens include variation, batch effects, and controlling for potential off-target effects. In this review, we discuss the efficacy of FUSION to identify novel inhibitors and oncogene-induced changes that may be cancer cell-specific as well as several potential pitfalls within FUSION and best practices to avoid them.

STEMI in Young Befogged by Aluminum Phosphide Toxicity-Role of ECMO as Salvage Therapy and Trimetazidine and Magnesium to Suppress Arrhythmias.

INTRODUCTION: Aluminum phosphide poisoning (ALP) has a high-mortality rate despite intensive care management, primarily because it causes severe myocardial depression. This case report highlights the subset of ALP patients presenting as ST elevation myocardial infarction (STEMI) with profound myocardial dysfunction and multiorgan failure and successfully treated with extracorporeal membrane oxygenation (ECMO), trimetazidine, and magnesium. CASE DESCRIPTION: A 25-year-old man without any comorbidities was brought to emergency department with dyspnea and hypotension. His electrocardiograph (ECG) revealed STEMI with elevated troponin levels, arterial blood gas (ABG) showed severe metabolic acidosis, and echocardiography (echo) revealed ejection fraction 15%. He was initiated on venoarterial (VA) ECMO in view of refractory hypotension. History of consumption of three tabs of celphos was revealed later by the family members. He progressed to cardiogenic shock, arrhythmias, respiratory failure, acute kidney injury with severe lactic acidosis, liver injury, pancreatitis, and disseminated intravascular coagulation (DIC). He was successfully supported by ECMO, hemodialysis, magnesium, trimetazidine, N-acetyl cysteine, inotropes, and blood products. He was weaned off ECMO on day 6 and was discharged home on day 12. Despite his severe and confounding clinical presentation, he had complete normalization of end-organ dysfunction with no neurological sequela. This case demonstrates the high index of suspicion required for ALP, given the potential for rapid progression and severe multiorgan toxicity. This report also highlights the importance of early referral to a tertiary care center with ECMO capability and also the role of magnesium and trimetazidine to suppress arrhythmias. CONCLUSION: Aluminum phosphide poisoning can present as STEMI with cardiogenic shock resulting in acute kidney injury, liver injury, pancreatitis, and DIC. Venoarterial ECMO provides an effective means of support until the recovery of organ function. Trimetazidine and magnesium are helpful in suppressing fatal arrhythmias. This report emphasizes that early recognition and early institution of ECMO can save many young lives who succumb to toxic effects of this poison. HOW TO CITE THIS ARTICLE: Rao CC, Himaaldev GJ. STEMI in Young Befogged by Aluminum Phosphide Toxicity-Role of ECMO as Salvage Therapy and Trimetazidine and Magnesium to Suppress Arrhythmias. Indian J Crit Care Med 2020;24(8):727-730.

PTAP motif duplication in the p6 Gag protein confers a replication advantage on HIV-1 subtype C.

HIV-1 subtype C (HIV-1C) may duplicate longer amino acid stretches in the p6 Gag protein, leading to the creation of an additional Pro-Thr/Ser-Ala-Pro (PTAP) motif necessary for viral packaging. However, the biological significance of a duplication of the PTAP motif for HIV-1 replication and pathogenesis has not been experimentally validated. In a longitudinal study of two different clinical cohorts of select HIV-1 seropositive, drug-naive individuals from India, we found that 8 of 50 of these individuals harbored a mixed infection of viral strains discordant for the PTAP duplication. Conventional and next-generation sequencing of six primary viral quasispecies at multiple time points disclosed that in a mixed infection, the viral strains containing the PTAP duplication dominated the infection. The dominance of the double-PTAP viral strains over a genetically similar single-PTAP viral clone was confirmed in viral proliferation and pairwise competition assays. Of note, in the proximity ligation assay, double-PTAP Gag proteins exhibited a significantly enhanced interaction with the host protein tumor susceptibility gene 101 (Tsg101). Moreover, Tsg101 overexpression resulted in a biphasic effect on HIV-1C proliferation, an enhanced effect at low concentration and an inhibitory effect only at higher concentrations, unlike a uniformly inhibitory effect on subtype B strains. In summary, our results indicate that the duplication of the PTAP motif in the p6 Gag protein enhances the replication fitness of HIV-1C by engaging the Tsg101 host protein with a higher affinity. Our results have implications for HIV-1 pathogenesis, especially of HIV-1C.