Introduction: Serine/threonine kinase 11 (LKB1/STK11) is one of the most mutated genes in NSCLC accounting for approximately one-third of cases and its activity is impaired in approximately half of KRAS-mutated NSCLC. At present, these patients cannot benefit from any specific therapy.Methods: Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type (WT) and KRASmutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both in vitro and in vivo.Results: In all the systems used here, the loss of LKB1 creates vulnerability and renders these cells particularly sensitive to ERK inhibitors both in vitro and in vivo. The same cells expressing a WT LKB1 poorly respond to these drugs. At the molecular level, in the absence of LKB1, ERK inhibitors induced a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect.Conclusions: This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS-mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Because ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing WT LKB1, predicts that treatment of LKB1mutated tumors with ERK inhibitors should have a favorable toxicity profile.

Non-Small-Cell Lung Cancer (NSCLC) is a poorly chemosensitive tumor and targeted therapies are only used for about 15% of patients where a specific driving and druggable lesion is observed (EGFR, ALK, ROS). KRAS is one of the most frequently mutated genes in NSCLC and patients harboring these mutations do not benefit from specific treatments. Sorafenib, a multi-target tyrosine kinase inhibitor, was proposed as a potentially active drug in KRAS-mutated NSCLC patients, but clinical trials results were not conclusive. Here we show that the NSCLC cells’ response to sorafenib depends on the type of KRAS mutation. KRAS G12V cells respond less to sorafenib than the wild-type counterpart, in vitro and in vivo. To overcome this resistance, we used high-throughput screening with a siRNA library directed against 719 human kinases, and Wee1 was selected as a sorafenib response modulator. Inhibition of Wee1 by its specific inhibitor MK1775 in combination with sorafenib restored the KRAS mutated cells’ response to the multi-target tyrosine kinase inhibitor. This combination of the Wee1 inhibitor with sorafenib, if confirmed in models with different genetic backgrounds, might be worth investigating further as a new strategy for KRAS mutated NSCLC.

IntroductionThe transcriptional factor NF-κB, composed by five subunits (RelA/p65, c-Rel, RelB, p50, p52), is largely involved in many facets of cellular physiology such as innate and adaptive immunity as well as inflammation. In addition, NF-kB play a central role in cancer cell survival and chemoresistance partly by its implication in cross-talks with redox-regulating proteins. Ferritin is the major iron storage protein; it is composed by a variable assembly of Heavy (FHC) and Light (FLC) subunits. FHC, in particular, has been widely demonstrated to be devoted in iron uptake and release thus controlling the redox homeostasis.Material and methodsK562 erythroleukemia cells were stably silenced for FHC by using the shRNA method. Then FHC reconstitution was achieved by transient transfection of a FHC specific expression vector. ROS were determined by incubating cells with the redox-sensitive probe 2‘−7‘-DCF. NAC was used to inhibit ROS production. MTT assay was performed to analyse cell viability. Increasing concentrations of Doxorubicin, ranging from 0 to 5 µM, were used to treat K562 cells.Results and discussionsThe results of this study highlighted that FHC amounts negatively affect NF-kB activation in K562 cells. FHC silencing was accompanied by an increased expression of the nuclear NF-kB subunit p65, FHC rescue determined nuclear p65 decrease. FHC silencing is responsible for intracellular ROS production and ROS are implicated in NF-kB pathway. To elucidate the relationship between ROS amount and nuclear p65 content, we determined ROS amounts in our in vitro model and evaluated p65 nuclear expression after treatment with the ROS scavenger NAC. First, we observed that, as expected, ROS levels increased upon FHC silencing and return to basal levels upon NAC treatment. Interestingly, NAC was also able to decrease nuclear p65 amount in FHC-silenced K562 cells. Considering the effect of NF-kB activated pathway on cell survival, we analysed the effect of FHC silencing-mediated p65 increase in K562 cells upon treatment with increasing doses of Doxorubicin. Cell viability assay highlighted that FHC-silencing was accompanied by an increased resistance to the drug with an IC50 about doubled compared to that of the K562 control cells at each the time points. This resistance of FHC-silenced cells was reverted upon NF-kB inhibitor transfection.ConclusionFHC silencing induced NF-kB activation in K562 cells through the modulation of intracellular ROS content. This regulatory axis can be used to modulate K562 chemoresistance.