Combining the SMAC mimetic LCL161 with Gemcitabine plus Cisplatin therapy inhibits and prevents the emergence of multidrug resistance in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly lethal gastrointestinal malignancy that has one of the worst prognoses among solid tumors. The combination of Gemcitabine + Cisplatin (GEM/CIS) remains the standard first-line treatment for advanced stage CCA. However, this drug combination yields only a modest objective response rate, and in cases that initially respond to this treatment, drug resistance commonly rapidly develops. To improve the efficiency of GEM/CIS therapy for CCA, a thorough understanding of the mechanism of GEM/CIS resistance in CCA is required. To that end - in this study, we developed several acquired GEM/CIS-resistant CCA cell lines and we screened those cell lines for acquired vulnerability. The screening process revealed that subset of CCA with GEM/CIS resistance acquired vulnerability to the small-molecule second mitochondrial-derived activator of caspases (SMAC) mimetics LCL161 and Birinapant. The observed acquired vulnerability was found to be associated with upregulation of an inhibitor of apoptosis protein 2 (cIAP2), a known target of SMAC mimetics. LCL161 or cIAP2-shRNA downregulated cIAP2 and restored the sensitivity to GEM/CIS in GEM/CIS-resistant CCA cell lines and in in vivo GEM/CIS-resistant xenograft models. A strong synergic effect was observed when LCL161 was added to GEM/CIS. Interestingly, this synergism was also observed in drug-naïve CCA cell lines, xenografts, and patient-derived organoids. This triplet therapy also prevented the emergence of multidrug-resistant CCA in in vitro and in vivo models. Our findings suggest that activation of cIAP2 allows CCA to escape GEM/CIS, and that suppression of cIAP2 reestablishes the apoptotic profile of CCA, thus restoring its vulnerability to GEM/CIS. The results of this study indicate that combining the SMAC mimetic LCL161 with GEM/CIS inhibits and prevents the emergence of multidrug resistance in CCA.

The Acquired Vulnerability Caused by CDK4/6 Inhibition Promotes Drug Synergism Between Oxaliplatin and Palbociclib in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult to treat cancers, and its nature of being largely refractory to most, if not all, current treatments results in generally poor prognosis and high mortality. Efficacious alternative therapies that can be used ubiquitously are urgently needed. Using acquired vulnerability screening, we observed that CCA cells that reprofile and proliferate under CDK4/6 inhibition became vulnerable to ribosomal biogenesis stress and hypersensitive to the anti-ribosome chemotherapy oxaliplatin. CCA cells overexpress the oncogenic ribosomal protein RPL29 under CDK4/6 inhibition in a manner that correlated with CDK4/6 inhibitor resistance. Depletion of RPL29 by small interfering RNAs (siRNAs) restored the sensitivity of CCA cells to CDK4/6 inhibition. Oxaliplatin treatment suppressed the RPL29 expression in the CDK4/6 inhibitor treated CCA cells and triggered RPL5/11-MDM2-dependent p53 activation and cancer apoptosis. In addition, we found that combination treatment with oxaliplatin and the CDK4/6 inhibitor palbociclib synergistically inhibited both parental and CDK4/6 inhibitor-resistant CCA, and prevented the emergence of CDK4/6 and oxaliplatin-resistant CCA. This drug combination also exerted suppressive and apoptosis effects on CCA in the in vitro 3-dimensional culture, patient-derived organoid, and in vivo xenograft CCA models. These results suggest the combination of the CDK4/6 inhibitor palbociclib and the anti-ribosome drug oxaliplatin as a potentially promising treatment for cholangiocarcinoma.

Corrigendum: Combining the SMAC mimetic LCL161 with Gemcitabine plus Cisplatin therapy inhibits and prevents the emergence of multidrug resistance in cholangiocarcinoma.

[This corrects the article DOI: 10.3389/fonc.2022.1021632.].

A novel bicyclic 2,4-diaminopyrimidine inhibitor of Streptococcus suis dihydrofolate reductase.

Streptococcus suis is a Gram-positive bacterial pathogen of pigs and an emerging zoonotic pathogen. It has become increasingly resistant to multiple classes of antibiotics. New drug candidates and knowledge of their targets are needed to combat antibiotic-resistant S. suis. In this study, the open-source Pathogen Box compound library was screened. Thirty hits that effectively inhibited S. suis growth at 10 µM were identified. Among the most potent hits, MMV675968 (a diaminoquinazoline analog) was shown to target S. suis dihydrofolate reductase (SsDHFR) via (1) growth inhibition of an E. coli surrogate whose growth is dependent on exogenously expressed SsDHFR and (2) inhibition of in vitro SsDHFR activity. Thymidine supplement is able to reverse growth inhibition by MMV675968 in both E. coli surrogate and S. suis, indicating that a thymidine-related pathway is a major target of MMV675968. Comparison of MMV675968 with seven DHFR inhibitors representing different core structures revealed that bicyclic 2,4-diaminopyrimidines with long and flexible side chains are highly effective in inhibiting SsDHFR and S. suis growth. MMV675968 and related compounds thus may serve as starting points for developing antibiotics against drug resistant S. suis.

Diaminoquinazoline MMV675968 from Pathogen Box inhibits Acinetobacter baumannii growth through targeting of dihydrofolate reductase.

Antibiotic resistance in Acinetobacter baumannii is a major global health threat. New drugs with novel chemical structures are needed to overcome a myriad of resistance mechanisms in A. baumannii. In this study, we screened an open-source Pathogen Box library for anti-A. baumannii compounds. Compound MMV675968 (a diaminoquinazoline analog) was the only non-reference compound found to inhibit the growth of all four A. baumannii test strains with IC50 of 0.6-2.7 µM, IC90 of 0.7-3.9 µM, and MIC of 1.6-10 µM. We showed that MMV675968 targeted A. baumannii dihydrofolate reductase (AbDHFR) as determined by an E. coli surrogate whose growth was dependent on AbDHFR function and by an in vitro DHFR activity assay. Additionally, chemical scaffolds of DHFR inhibitors that are effective as antibiotics against A. baumannii were identified using an in vitro DHFR activity assay and A. baumannii growth inhibition. MMV675968 was the most potent among DHFR inhibitors tested in inhibiting A. baumannii growth. This study shows for the first time that MMV675968 inhibits A. baumannii growth via selective inhibition of AbDHFR and is therefore a promising scaffold for further antibiotic development against A. baumannii.