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The mating types of 124 and 88 single spore isolates, isolated at random from Volvariella volvacea strains V23 ( A mating type locus alleles A1 + A2 ) and PY ( A mating type locus alleles A3 + A4 ) respectively, were determined by molecular screening using four primer pairs designed according to the sequences of the A mating type genes ( HD1 and HD2 ) of the two parental strains. Of the single spore isolates from strain V23, 101were confirmed as homokaryons, 35 of which were mating type A1 and 66 mating type A2 . The remaining 23 were heterokaryons carrying both A1 and A2 mating type loci. Of the single spore isolates from strain PY, 72 were confirmed as homokaryons, of which 41 were mating type A3 and 31 mating type A4 . Sixteen were heterokaryons carrying both A3 and A4 mating type loci. Cross-breeding between 72 compatible pairs generated 58 hybrids, three of which were designated high quality hybrids based on agronomic traits determined by cultivation experiments. Our research has established that the molecular marker-assisted cross-breeding technique represents a practical and highly efficient system for generating improved V. volvacea cultivars. [ABSTRACT FROM AUTHOR]

Background: Tyrosine kinase inhibitors (TKIs) are mainstays of cancer treatment. However, their clinical benefits are often constrained by acquired resistance. To overcome such outcomes, we have rationally engineered APG-2449 as a novel multikinase inhibitor that is highly potent against oncogenic alterations of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), and focal adhesion kinase (FAK). Here we present the preclinical evaluation of APG-2449, which exhibits antiproliferative activity in cells carrying ALK fusion or secondary mutations.Methods: KINOMEscan® and LANCE TR-FRET were used to characterize targets and selectivity of APG-2449. Water-soluble tetrazolium salt (WST-8) viability assay and xenograft tumorigenicity were employed to evaluate therapeutic efficacy of monotherapy or drug combination in preclinical models of solid tumors. Western blot, pharmacokinetic, and flow cytometry analyses, as well as RNA sequencing were used to explore pharmacokinetic-pharmacodynamic correlations and the mechanism of actions driving drug combination synergy.Results: In mice bearing wild-type or ALK/ROS1-mutant non-small-cell lung cancer (NSCLC), APG-2449 demonstrates potent antitumor activity, with correlations between pharmacokinetics and pharmacodynamics in vivo. Through FAK inhibition, APG-2449 sensitizes ovarian xenograft tumors to paclitaxel by reducing CD44+ and aldehyde dehydrogenase 1-positive (ALDH1+) cancer stem cell populations, including ovarian tumors insensitive to carboplatin. In epidermal growth factor receptor (EGFR)-mutated NSCLC xenograft models, APG-2449 enhances EGFR TKI-induced tumor growth inhibition, while the ternary combination of APG-2449 with EGFR (osimertinib) and mitogen-activated extracellular signal-regulated kinase (MEK; trametinib) inhibitors overcomes osimertinib resistance. Mechanistically, phosphorylation of ALK, ROS1, and FAK, as well as their downstream components, is effectively inhibited by APG-2449.Conclusions: Taken together, our studies demonstrate that APG-2449 exerts potent and durable antitumor activity in human NSCLC and ovarian tumor models when administered alone or in combination with other therapies. A phase 1 clinical trial has been initiated to evaluate the safety and preliminary efficacy of APG-2449 in patients with advanced solid tumors, including ALK+ NSCLC refractory to earlier-generation ALK inhibitors.Trial Registration: Clinicaltrial.gov registration: NCT03917043 (date of first registration, 16/04/2019) and Chinese clinical trial registration: CTR20190468 (date of first registration, 09/04/2019). [ABSTRACT FROM AUTHOR]