Kaposi's Sarcoma-Associated Herpesvirus Viral Interleukin-6 Signaling Upregulates Integrin ß3 Levels and Is Dependent on STAT3.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of two B-cell lymphoproliferative diseases and Kaposi's sarcoma, an endothelial-cell-driven cancer. KSHV viral interleukin-6 (vIL-6) is a viral homolog of human IL-6 (hIL-6) that is expressed in KSHV-associated malignancies. Previous studies have shown that the expression of the integrin ß3 (ITGB3) subunit is induced upon KSHV infection. Here we report that KSHV vIL-6 is able to induce the expression of ITGB3 and increase surface expression of the αVß3 integrin heterodimer. We demonstrated using small interfering RNA (siRNA) depletion and inhibitor studies that KSHV vIL-6 can increase ITGB3 by inducing STAT3 signaling. Furthermore, we found that secreted vIL-6 is capable of inducing ITGB3 in endothelial cells in a paracrine manner. Importantly, the ability to induce ITGB3 in endothelial cells seems to be specific to vIL-6, as overexpression of hIL-6 alone did not affect levels of this integrin. Our lab and others have previously shown that vIL-6 can induce angiogenesis, and we investigated whether ITGB3 was involved in this process. We found that siRNA depletion of ITGB3 in vIL-6-expressing endothelial cells resulted in a decrease in adhesion to extracellular matrix proteins. Moreover, depletion of ITGB3 hindered the ability of vIL-6 to promote angiogenesis. In conclusion, we found that vIL-6 can singularly induce ITGB3 and that this induction is dependent on vIL-6 activation of the STAT3 signaling pathway.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies: multicentric Castleman's disease, primary effusion lymphoma, and Kaposi's sarcoma. Kaposi's sarcoma is a highly angiogenic tumor that arises from endothelial cells. It has been previously reported that KSHV infection of endothelial cells leads to an increase of integrin αVß3, a molecule observed to be involved in the angiogenic process of several malignancies. Our data demonstrate that the KSHV protein viral interleukin-6 (vIL-6) can induce integrin ß3 in an intracellular and paracrine manner. Furthermore, we showed that this induction is necessary for vIL-6-mediated cell adhesion and angiogenesis, suggesting a potential role of integrin ß3 in KSHV pathogenesis and development of Kaposi's sarcoma.

Amivantamab efficacy in wild-type EGFR NSCLC tumors correlates with levels of ligand expression.

Amivantamab is an FDA-approved bispecific antibody targeting EGF and Met receptors, with clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Amivantamab efficacy has been demonstrated to be linked to three mechanisms of action (MOA): immune cell-mediated killing, receptor internalization and degradation, and inhibition of ligand binding to both EGFR and Met receptors. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in wild-type (WT) EGFR (EGFRWT) NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers. Treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab or with an AREG-targeting antibody inhibited ligand-induced signaling and cell/tumor proliferation/growth. Across 11 EGFRWT NSCLC patient-derived xenograft models, amivantamab efficacy correlated with AREG RNA levels. Interestingly, in these models, amivantamab anti-tumor activity was independent of Fc engagement with immune cells, suggesting that, in this context, the ligand-blocking function is sufficient for amivantamab maximal efficacy. Finally, we demonstrated that in lung adenocarcinoma patients, high expression of AREG and EGFR mutations were mutually exclusive. In conclusion, these data 1) highlight EGFR ligand AREG as a driver of tumor growth in some EGFRWT NSCLC models, 2) illustrate the preclinical efficacy of amivantamab in ligand-driven EGFRWT NSCLC, and 3) identify AREG as a potential predictive biomarker for amivantamab activity in EGFRWT NSCLC.

Kaposi's sarcoma-associated herpesvirus viral protein kinase augments cell survival.

Oncogenic viruses have developed various strategies to antagonize cell death and maintain lifelong persistence in their host, a relationship that may contribute to cancer development. Understanding how viruses inhibit cell death is essential for understanding viral oncogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three different cancers in the human population, including Kaposi's sarcoma (KS), the most common cancer in HIV patients. Previous studies have indicated that the KSHV-encoded viral protein kinase (vPK) impacts many processes dysregulated in tumorigenesis. Here, we report that vPK protects cells from apoptosis mediated by Caspase-3. Human umbilical vein endothelial cells (HUVECs) expressing vPK (HUVEC-vPK) have a survival advantage over control HUVEC under conditions of extrinsic- and intrinsic-mediated apoptosis. Abolishing the catalytic activity of vPK attenuated this survival advantage. We found that KSHV vPK-expressing HUVECs exhibited increased activation of cellular AKT kinase, a cell survival kinase, compared to control cells without vPK. In addition, we report that vPK directly binds the pleckstrin homology (PH) domain of AKT1 but not AKT2 or AKT3. Treatment of HUVEC-vPK cells with a pan-AKT inhibitor Miransertib (ARQ 092) reduced the overall phosphorylation of AKT, resulting in the cleavage of Caspase-3 and the induction of apoptosis. Furthermore, vPK expression activated VEGF/VEGFR2 in HUVECs and promoted angiogenesis through the AKT pathway. vPK expression also inhibited the cytotoxicity of cisplatin in vitro and in vivo. Collectively, our findings demonstrate that vPK's ability to augment cell survival and promote angiogenesis is critically dependent on AKT signaling, which is relevant for future therapies for treating KSHV-associated cancers.

Combined Inhibition of Akt and mTOR Is Effective Against Non-Hodgkin Lymphomas.

Non-Hodgkin lymphoma (NHL) are a diverse group of hematological malignancies comprised of over 60 subtypes. These subtypes range from indolent to aggressive. The PI3K/Akt/mTOR pathway has been shown to contribute to cell survival and proliferation and is constitutively active in most NHL. MK-7075 (miransertib) and MK-4440 are small molecules that effectively inhibit Akt and have entered clinical development. Using in vitro and in vivo models of NHL, we explored targeting the kinase Akt with miransertib and MK-4440 alone or in combination with the mTORC1 inhibitor, rapamycin (sirolimus). Both Akt inhibitors inhibited the pathway and NHL proliferation in a subtype-dependent manner. However, these compounds had a minimal effect on the viability of primary B-cells. Importantly, the combination of miransertib and sirolimus synergistically reduced cell proliferation in NHL, including in one indolent subtype, e.g., follicular lymphoma (FL), and two aggressive subtypes, e.g., diffuse large B-cell lymphoma (DLBCL) and primary effusion lymphoma (PEL). To establish in vivo efficacy, we used several xenograft models of FL, DLBCL, and PEL. The results obtained in vivo were consistent with the in vitro studies. The FL xenograft was highly sensitive to the inhibition of Akt alone; however, the tumor burden of PEL xenografts was only significantly reduced when both Akt and mTORC1 were targeted. These data suggest that targeting the PI3K/Akt/mTOR pathway with Akt inhibitors such as miransertib in combination with mTOR inhibitors serves as a broadly applicable therapeutic in NHL.

High-Density Amplicon Sequencing Identifies Community Spread and Ongoing Evolution of SARS-CoV-2 in the Southern United States.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constantly evolving. Prior studies focused on high-case-density locations, such as the northern and western metropolitan areas of the United States. This study demonstrates continued SARS-CoV-2 evolution in a suburban southern region of the United States by high-density amplicon sequencing of symptomatic cases. 57% of strains carry the spike D614G variant, which is associated with higher genome copy numbers, and its prevalence expands with time. Four strains carry a deletion in a predicted stem loop of the 3' UTR. The data are consistent with community spread within local populations and the larger continental United States. The data instill confidence in current testing sensitivity and validate "testing by sequencing" as an option to uncover cases, particularly nonstandard coronavirus disease 2019 (COVID-19) clinical presentations. This study contributes to the understanding of COVID-19 through an extensive set of genomes from a non-urban setting and informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate in the United States.

Modulation of Angiogenic Processes by the Human Gammaherpesviruses, Epstein-Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus.

Angiogenesis is the biological process by which new blood vessels are formed from pre-existing vessels. It is considered one of the classic hallmarks of cancer, as pathological angiogenesis provides oxygen and essential nutrients to growing tumors. Two of the seven known human oncoviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), belong to the Gammaherpesvirinae subfamily. Both viruses are associated with several malignancies including lymphomas, nasopharyngeal carcinomas, and Kaposi's sarcoma. The viral genomes code for a plethora of viral factors, including proteins and non-coding RNAs, some of which have been shown to deregulate angiogenic pathways and promote tumor growth. In this review, we discuss the ability of both viruses to modulate the pro-angiogenic process.

Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy.

Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy.