using-rna-sequencing-to-detect-novel-splice-variants-related-to-drug

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in <em>In Vitro</em> Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

VU University medical center

Current cancer research is being increasingly focused on the study of distinctive characters of tumour metabolism, resulting in a switch from oxidative phosphorylation to glycolysis (Warburg effect). Isoform 5 of human lactate dehydrogenase (hLDH5), which catalyzes the final step in the glycolytic cascade (pyruvate to lactate), constitutes a relatively new and untapped anti-cancer target. In this study, careful design and synthesis of a selected series of aryl-substituted N-hydroxyindole-2-carboxylates (NHIs) has led to several hLDH5-inhibitors, showing "first-in-class" potency and isoform selectivity. Enzyme kinetics studies indicated that these inhibitors exhibit a competitive mode of inhibition. Some representative examples were tested against two human pancreatic carcinoma cell lines, and displayed a good anti-proliferative activity, which was even more evident under hypoxic conditions.

N-Hydroxyindole-based inhibitors of lactate dehydrogenase against cancer cell proliferation.

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.

Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery.

Malignant pleural mesothelioma (MPM) is a lethal disease with scarce therapeutic options, and preclinical studies on new targeted-agents are warranted. Because previous studies reported high c-Met expression and alterations in the microtubules network in most MPM samples, we evaluated the activity of tivantinib, which has been recently suggested to affect microtubule polymerization in addition to inhibiting c-Met. In four MPM cell lines tivantinib inhibited both c-Met activity and microtubule polymerization, resulting in inhibition of cell-growth with IC50s ranging between 0.3 µM (MSTO-211H) and 2.4 µM (H2052). Furthermore tivantinib synergistically enhanced the antiproliferative and proapoptotic activity of pemetrexed, as detected by sulforhodamine-B-assay and flow cytometry. The synergistic interaction was associated with reduction of thymidylate synthase expression and inhibition of migratory activity. In aggregate, these data show the ability of tivantinib to specifically target key pathways in MPM cells and synergistically interact with pemetrexed, supporting further studies on this therapeutic approach.

Synergistic activity of the c-Met and tubulin inhibitor tivantinib (ARQ197) with pemetrexed in mesothelioma cells.

The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.

NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor.

The association of aberrant folylpolyglutamate synthetase splicing with ex vivo methotrexate resistance and clinical outcome in childhood acute lymphoblastic leukemia.

Article	Year
N-Hydroxyindole-based inhibitors of lactate dehydrogenase against cancer cell proliferation. European journal of medicinal chemistry\| PubMed ID: 21944286	2011
Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery. Nature biotechnology\| PubMed ID: 24633243	2014
Synergistic activity of the c-Met and tubulin inhibitor tivantinib (ARQ197) with pemetrexed in mesothelioma cells. Current drug targets\| PubMed ID: 25483224	2014
NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor. Oncotarget\| PubMed ID: 26015408	2015
The association of aberrant folylpolyglutamate synthetase splicing with ex vivo methotrexate resistance and clinical outcome in childhood acute lymphoblastic leukemia. Haematologica\| PubMed ID: 27036162	2016

Rocco Sciarrillo

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