Relationship between the transcriptional expression of PIM1 and local control in patients with head and neck squamous cell carcinomas treated with radiotherapy

Purpose Proviral integration site for Moloney murine leukemia virus (PIMs) are proto-oncogenes encoding serine/threonine kinases that phosphorylate a variety of substrates involved in the regulation of cellular processes. Elevated expression of PIM-1 has been associated with poor prognosis in several types of cancer. There are no studies that have analyzed the response to radiotherapy in patients with head and neck squamous cell carcinoma (HNSCC) according to the expression of PIM-1. The aim of our study was to analyze the relationship between the transcriptional expression of PIM-1 and local response to radiotherapy in HNSCC patients. Methods We determined the transcriptional expression of PIM-1 in 135 HNSCC patients treated with radiotherapy, including patients treated with chemoradiotherapy (n = 65) and bioradiotherapy (n = 15). Results During the follow-up, 48 patients (35.6%) had a local recurrence of the tumor. Patients with local recurrence had a higher level of PIM-1 expression than those who achieved local control of the disease (P = 0.017). Five-year local recurrence-free survival for patients with a high expression of PIM-1 (n = 43) was 44.6% (95% CI 29.2–60.0%), and for patients with low expression (n = 92) it was 71.9% (95% CI 62.5–81.3%) (P = 0.007). According to the results of multivariate analysis, patients with a high PIM-1 expression had a 2.2-fold increased risk of local recurrence (95% CI 1.22–4.10, P = 0.009). Conclusion Patients with elevated transcriptional expression levels of PIM-1 had a significantly higher risk of local recurrence after radiotherapy.

New Approaches to Multi-Parametric HIV-1 Genetics Using Multiple Displacement Amplification: Determining the What, How, and Where of the HIV-1 Reservoir

Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.

Dibenzofuran Derivatives Inspired from Cercosporamide as Dual Inhibitors of Pim and CLK1 Kinases

Pim kinases (proviral integration site for Moloney murine leukemia virus kinases) are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Thus, Pim kinases are validated as targets for antitumor therapy. In this context, our combined efforts in natural product-inspired library generation and screening furnished very promising dibenzo[b,d]furan derivatives derived from cercosporamide. Among them, lead compound 44 was highlighted as a potent Pim-1/2 kinases inhibitor with an additional nanomolar IC50 value against CLK1 (cdc2-like kinases 1) and displayed a low micromolar anticancer potency towards the MV4-11 (AML) cell line, expressing high endogenous levels of Pim-1/2 kinases. The design, synthesis, structure–activity relationship, and docking studies are reported herein and supported by enzyme, cellular assays, and Galleria mellonella larvae testing for acute toxicity.

PIM kinases mediate resistance to cisplatin chemotherapy in hepatoblastoma

Despite increasing incidence, treatment for hepatoblastoma has not changed significantly over the past 20 years. Chemotherapeutic strategies continue to rely on cisplatin, as it remains the most active single agent against hepatoblastoma. However, chemoresistance remains a significant challenge with 54–80% of patients developing resistance to chemotherapy after 4–5 cycles of treatment. Stem cell-like cancer cells (SCLCCs) are a subset of cells thought to play a role in chemoresistance and disease recurrence. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, specifically PIM3, play a role in hepatoblastoma cell proliferation and tumor growth and maintain the SCLCC phenotype. Here, we describe the development of a cisplatin-resistant hepatoblastoma xenograft model of the human HuH6 cell line and a patient-derived xenograft, COA67. We provide evidence that these cisplatin-resistant cells are enriched for SCLCCs and express PIM3 at higher levels than cisplatin-naïve cells. We demonstrate that PIM inhibition with AZD1208 sensitizes cisplatin-resistant hepatoblastoma cells to cisplatin, enhances cisplatin-mediated apoptosis, and decreases the SCLCC phenotype seen with cisplatin resistance. Together, these findings indicate that PIM inhibition may be a promising adjunct in the treatment of hepatoblastoma to effectively target SCLCCs and potentially decrease chemoresistance and subsequent disease relapse.

Selectivity and Potency of Natural Product Pim Kinase Inhibitors Identified By in Silico Docking

PIM3 (Proviral Integration site for Maloney murine leukemia virus kinase 3) is a proto-oncogene with serine/threonine kinase activity that prevents apoptosis, promotes cell survival, and stimulates protein translation. Additionally, PIM3 functions in inflammation and immunity pathways. PIM3 inhibitors are being developed to treat cancer and inflammation-related disorders. Here we screen a 98,000 compound virtual library of natural products to identify those that are predicted to fit in the ATP site of PIM3. Since the structure of PIM3 has not been determined experimentally, we performed molecular structure prediction using the SWISS-MODEL tool to generate a PIM3 model structure for in silico screening. Compounds predicted to fit the ATP binding site of PIM3 were validated using biochemical assays, revealing activity against PIM3 for all 8 candidates, with potencies mostly in the micromolar range. We tested several analogs of two validated candidates, the diosgenin glycoside dioscin and the biflavonoid hinokiflavone. Among five dioscin analogs, three exhibit similar potency against PIM3, and with some selectivity for PIM3 versus PIM1 and 2. Meanwhile, 3 of seven biflavonoid analogs exhibit sub-micromolar IC50 potency against PIM3, but with less selectivity for PIM3 versus PIM 1 and 2.

New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design, Synthesis and Biological Evaluation

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure–activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

Relationship between CD4 T cell turnover, cellular differentiation and HIV persistence during ART

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.

HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus

BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred for non-suppressible viremia (plasma HIV-1 RNA above 40 copies/ml) who reported adherence to ART and did not show drug resistance to their current regimen.METHODSSamples were collected from at least two time points from eight donors who had non-suppressible viremia for more than six months on ART. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were PCR-amplified and sequenced for evidence of clones of cells that produced infectious viruses. Clones were identified by host-proviral integration site analysis.RESULTSHIV-1 genomic RNAs with identical sequences were identified in plasma samples from all eight donors. The identical viral RNA sequences did not change over time and lacked resistance to the ART regimen. In four of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication-competent. Integration sites for infectious proviruses from those four donors were mapped to introns of the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. The sizes of the clones were from 50 million to 350 million cells.CONCLUSIONClones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia despite medication adherence and absence of drug resistance. The mechanisms involved in clonal expansion and persistence need to be defined to eliminate viremia and the HIV-1 reservoir.

Insights into the Interaction Mechanisms of the Proviral Integration Site of Moloney Murine Leukemia Virus (Pim) Kinases with Pan-Pim Inhibitors PIM447 and AZD1208: A Molecular Dynamics Simulation and MM/GBSA Calculation Study

Based on the up-regulation of the proviral integration site of the Moloney murine leukemia virus (Pim) kinase family (Pim1, 2, and 3) observed in several types of leukemias and lymphomas, the development of pan-Pim inhibitors is an attractive therapeutic strategy. While only PIM447 and AZD1208 have entered the clinical stages. To elucidate the interaction mechanisms of three Pim kinases with PIM447 and AZD1208, six Pim/ligand systems were studied by homology modeling, molecular docking, molecular dynamics (MD) simulation and molecular mechanics/generalized Born surface area (MM/GBSA) binding free energy calculation. The residues of the top group (Leu44, Val52, Ala65, Lys67, and Leu120 in Pim1) dominated the pan-Pim inhibitors binding to Pim kinases. The residues of the bottom group (Gln127, Asp128, and Leu174 in Pim1) were crucial for Pims/PIM447 systems, while the contributions of these residues were decreased sharply for Pims/AZD1208 systems. It is likely that the more potent pan-Pim inhibitors should be bound strongly to the top and bottom groups. The residues of the left, right and loop groups were located in the loop regions of the binding pocket, however, the flexibility of these regions triggered the protein interacting with diverse pan-Pim inhibitors efficiently. We hope this work can provide valuable information for the design of novel pan-Pim inhibitors in the future.