Acquired Resistance Mutations to EGFR Treatment in Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is currently the number one cause of cancer death in the United States for both men and women.1 Mutations in the epidermal growth factor receptor (EGFR) gene are detected in approximately 30% of individuals with advanced NSCLC in Asia and 10-15% in Western countries.2 For patients harboring activating EGFR mutations, treatment includes the use of first or second-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs), such as afatinib, gefitinib, or erlotinib. The purpose of this case study is to review the pathophysiology of the progression of NSCLC in a 63-year-old non-smoking Caucasian woman. The patient presented with worsening back pain for four months, sudden onset of lower extremity weakness, and unintentional 20-pound weight loss. Imaging revealed spinal cord compression, a right upper lung mass with hilar adenopathy, multiple vertebral metastases, adrenal lesions, and a mass in the left lobe of the liver. Transbronchial biopsy of the lung mass in the patient confirmed NSCLC of the adenocarcinoma type. Next Generation Sequencing (NGS) identified an L858R mutation in exon 21 of EGFR, which results in activation of the tyrosine kinase (TK) domain of the EGFR protein product, without the need for ligand binding and a decreased binding affinity for ATP. The patient began daily erlotinib therapy with subsequent regression of disease at four months follow-up. After 12 months on erlotinib the patient developed radiographic progression with a T790M mutation in exon 20, the most common resistance mutation secondary to treatment with first generation TKIs. The patient was subsequently started on the third-generation TKI, osimertinib. After 12 months of treatment the patient developed the EGFR C797S tertiary mutation leading to osimertinib resistance and further progression of the disease. According to clinical guidelines, all patients with non-squamous NSCLC should be tested for mutations in EGFR. This case study serves as evidence that constant monitoring of EGFR positive patients is essential, as there are multiple ways in which cells develop resistance to TKI treatment.

The meiosis-specific Cdc20 family-member Ama1 promotes binding of the Ssp2 activator to the Smk1 MAP kinase.

Smk1 is a meiosis-specific MAP kinase (MAPK) in budding yeast that is required for spore formation. It is localized to prospore membranes (PSMs), the structures that engulf haploid cells during meiosis II (MII). Similar to canonically activated MAPKs, Smk1 is controlled by phosphorylation of its activation-loop threonine (T) and tyrosine (Y). However, activation loop phosphorylation occurs via a noncanonical two-step mechanism in which 1) the cyclin-dependent kinase activating kinase Cak1 phosphorylaytes T207 during MI, and 2) Smk1 autophosphorylates Y209 as MII draws to a close. Autophosphorylation of Y209 and catalytic activity for substrates require Ssp2, a meiosis-specific protein that is translationally repressed until anaphase of MII. Ama1 is a meiosis-specific targeting subunit of the anaphase-promoting complex/cyclosome that regulates multiple steps in meiotic development, including exit from MII. Here, we show that Ama1 activates autophosphorylation of Smk1 on Y209 by promoting formation of the Ssp2/Smk1 complex at PSMs. These findings link meiotic exit to Smk1 activation and spore wall assembly

Autophosphorylation of the Smk1 MAPK is spatially and temporally regulated by Ssp2 during meiotic development in yeast.

Smk1 is a meiosis-specific MAPK that controls spore wall morphogenesis in Saccharomyces cerevisiae. Although Smk1 is activated by phosphorylation of the threonine (T) and tyrosine (Y) in its activation loop, it is not phosphorylated by a dual-specificity MAPK kinase. Instead, the T is phosphorylated by the cyclin-dependent kinase (CDK)-activating kinase, Cak1. The Y is autophosphorylated in an intramolecular reaction that requires a meiosis-specific protein named Ssp2. The meiosis-specific CDK-like kinase, Ime2, was previously shown to positively regulate Smk1. Here we show that Ime2 activity is required to induce the translation of SSP2 mRNA at anaphase II. Ssp2 protein is then localized to the prospore membrane, the structure where spore wall assembly takes place. Next the carboxy-terminal portion of Ssp2 forms a complex with Smk1 and stimulates the autophosphorylation of its activation-loop Y residue. These findings link Ime2 to Smk1 activation through Ssp2 and define a developmentally regulated mechanism for activating MAPK at specific locations in the cell

Combining genomic and epidemiological data to compare the transmissibility of SARS-CoV-2 variants Alpha and Iota.

SARS-CoV-2 variants shaped the second year of the COVID-19 pandemic and the discourse around effective control measures. Evaluating the threat posed by a new variant is essential for adapting response efforts when community transmission is detected. In this study, we compare the dynamics of two variants, Alpha and Iota, by integrating genomic surveillance data to estimate the effective reproduction number (Rt) of the variants. We use Connecticut, United States, in which Alpha and Iota co-circulated in 2021. We find that the Rt of these variants were up to 50% larger than that of other variants. We then use phylogeography to show that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of Alpha were larger than those resulting from Iota introductions. By monitoring the dynamics of individual variants throughout our study period, we demonstrate the importance of routine surveillance in the response to COVID-19

Regulation of Non-Canonical MAP Kinase Signaling

Mitogen activated protein kinases (MAPKs) are signaling enzymes that control diverse processes in eukaryotic cells including proliferation, differentiation, and development. Many MAPKs are activated through well-studied canonical signaling pathways in which a MAPK kinase phosphorylates a T-X-Y motif in the activation loop of the MAPK. However, MAPKs can also be activated by non-canonical mechanisms. Smk1 is a meiosis-specific MAPK in the yeast S. cerevisiae that controls spore wall morphogenesis. The Smk1 pathway provides a model system to interrogate mechanisms of non-canonical MAPK signaling in the context of a developmental program. Previous genetic studies identified CAK1, AMA1 , IME2 and SSP2 as genes that regulate Smk1. Cak1 is a cyclin-dependent kinase (CDK)-activating kinase. Ama1 is a meiosis-specific targeting factor of the APC/C E3 ubiquitin ligase that controls exit from meiosis II (MII). Ime2 is a meiosis-specific CDK-like kinase. Ssp2 is a meiosis-specific protein required for spore morphogenesis, whose biochemical function has not previously been described. In this thesis we provide evidence for a novel spatiotemporal regulatory mechanism in which Cak1 phosphorylates Smk1 on its activation-loop T (T207) as the meiotic divisions are taking place. Smk1 autophosphorylates its activation-loop Y (Y209) as Ssp2 binds Smk1 at membranes that surround haploid products (prospore membranes; PSMs) during exit from meiosis II. Ssp2/Smk1 complex formation is regulated through multiple mechanisms, preventing premature MAPK activity. First, although both SMK1 and SSP2 are transcriptionally induced as cells enter meiosis I (MI), SMK1 mRNA is translated almost immediately, while SSP2 mRNA is translationally repressed until Ime2 triggers its derepression at MII. Next, Ssp2 localizes to PSMs, but inhibitory phosphates prevent it from binding Smk1. These phosphates are removed during meiotic exit, as PSMs close around each meiotic product in a step that requires Ama1. Finally, dephosphorylated Ssp2 binds Smk1, inducing autophosphorylation, thus coupling meiotic exit to Smk1 activation. We also show that RRM-like domains in the C-terminus of Ssp2 bind Smk1. Taken together, this work has defined novel mechanisms for spatiotemporally regulating the Smk1 MAPK during meiotic development

Functional interrogation of Lynch syndrome-associated MSH2 missense variants via CRISPR-Cas9 gene editing in human embryonic stem cells.

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in-cellulo functional assay in which we engineered site-specific MSH2 VUS using clustered regularly interspaced short palindromic repeats-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell-based assay system for functional testing of MMR gene VUS will facilitate the identification of high-risk LS patients

Molecular profiling of gynecologic cancers for treatment and management of disease - demonstrating clinical significance using the AMP/ASCO/CAP guidelines for interpretation and reporting of somatic variants.

Molecular features of gynecologic cancers have been investigated in comprehensive studies, but correlation of these molecular signatures with clinical significance for precision medicine is yet to be established. Towards this end, we evaluated 95 gynecologic cancer cases submitted for testing using The JAX ActionSeq™ NGS panel. Molecular profiles were studied and compared to TCGA datasets to identify similarities and distinguishing features among subtypes. We identified 146 unique clinically significant variants (Tier I and II) across 45 of the 212 genes (21%), in 87% (83/95) of cases. TP53, PTEN, ARID1A, PIK3CA and ATM were the most commonly mutated genes; CCNE1 and ERBB2 amplifications were the most frequently detected copy-number alterations. PARP inhibitors were among the most commonly reported drug class with clinical trials, consistent with the frequency of DNA damage-response pathway mutations in our cohort. Overall, our study provides additional insight into the molecular profiles of gynecologic cancers, highlighting regulatory pathways involved, raising the potential implications for targeted therapeutic options currently available

Molecular profiling of CNS tumors for the treatment and management of disease.

The World Health Organization (WHO) has defined more than 130 distinct central nervous system (CNS) tumor entities, of which glioblastoma is the most fatal primary brain tumor. However, the correlation of the molecular signatures of glioblastoma with clinical significance for precision medicine is not well-known. How, and to what extent these variants may affect clinical decision making remains uncertain. Here, we evaluate 48 glioblastomas submitted for testing using the JAX ActionSeq™ Next-generation sequencing (NGS) panel. We identified 131 clinically significant variants (Tier I and II) across 30 of the 212 genes (14%). TP53, EGFR, PTEN, IDH1 were the most commonly mutated genes; EGFR, CDK4 amplifications, and CDKN2A deletion were the most frequently detected copy-number alterations. CDK4/6 and PI3K inhibitors were among the most commonly reported drug class with FDA approved therapies and investigational therapies, which is consistent with the frequencies of these genes in our cohort. Overall, our study established the molecular profiles of glioblastoma based on the 2017 joint consensus guidelines by AMP/ASCO/CAP and provides the potential implications for targeted therapeutic options currently available