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.

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.].

Premature Senescence and Telomere Shortening Induced by Oxidative Stress From Oxalate, Calcium Oxalate Monohydrate, and Urine From Patients With Calcium Oxalate Nephrolithiasis.

Oxidative stress, a well-known cause of stress-induced premature senescence (SIPS), is increased in patients with calcium oxalate (CaOx) kidney stones (KS). Oxalate and calcium oxalate monohydrate (COM) induce oxidative stress in renal tubular cells, but to our knowledge, their effect on SIPS has not yet been examined. Here, we examined whether oxalate, COM, or urine from patients with CaOx KS could induce SIPS and telomere shortening in human kidney (HK)-2 cells, a proximal tubular renal cell line. Urine from age- and sex-matched individuals without stones was used as a control. In sublethal amounts, H2O2, oxalate, COM, and urine from those with KS evoked oxidative stress in HK-2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity, but urine from those without stones did not. The proportion of senescent HK-2 cells, as indicated by SA-ßgal staining, increased after treatment with H2O2, oxalate, COM, and urine from those with KS. Expression of p16 was higher in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than it was in cells treated with urine from those without stones and untreated controls. p16 was upregulated in the SA-ßgal positive cells. Relative telomere length was shorter in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than that in cells treated with urine from those without stones and untreated controls. Transcript expression of shelterin components (TRF1, TRF2 and POT1) was decreased in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS, in which case the expression was highest. Urine from those without KS did not significantly alter TRF1, TRF2, and POT1 mRNA expression in HK-2 cells relative to untreated controls. In conclusion, oxalate, COM, and urine from patients with CaOx KS induced SIPS and telomere shortening in renal tubular cells. SIPS induced by a lithogenic milieu may result from upregulation of p16 and downregulation of shelterin components, specifically POT1, and might contribute, at least in part, to the development of CaOx KS.

Clinical validation of urinary indole-reacted calcium oxalate crystallization index (iCOCI) test for diagnosing calcium oxalate urolithiasis.

An indole-reacted calcium oxalate crystallization index (iCOCI) test was developed to quantify the total competence of urine to precipitate calcium oxalate (CaOx) crystals. We conducted the prospective cohort study in accordance with the STARD guideline to evaluate the accuracy of urinary iCOCI test (index test) for diagnosing urolithiasis. A total of 281 participants were recruited for the study. Levels of urinary iCOCI were determined in the pre-diagnostic 24-h urine samples. Positive urinary iCOCI (≥ 0.6 COM eqv., g/L) was accounted for 51% (144/281), and the rest of 49% (137/281) were negative. Non-contrast CT imaging (reference standard) was subsequently performed for the definite diagnosis of urolithiasis to divide the participants into two groups, non-stone subjects (NSS, n = 122) and stone-forming subjects (SFS, n = 159). It should be noted that only subjects who currently had urinary stone at the time of study were classified as SFS. Urinary iCOCI levels in the SFS were significantly higher than the NSS. ROC analysis revealed an area under curve (AUC) of 0.893 (95% CI: 0.855-0.932) in separating NSS from all SFS. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, positive likelihood ratio (LH+) and negative likelihood ratio (LH-) of urinary iCOCI test for diagnosis of all urolithiasis were 87%, 80%, 84%, 84%, 83%, 4.44 and 0.16, respectively. Of 159 SFS, 38 were confirmed to have CaOx stones. Among these 38 CaOx SFS, only 2 had negative urinary iCOCI test. The AUC of urinary iCOCI test for separating CaOx SFS from NSS was markedly high (0.946, 95% CI: 0.914-0.978). Sensitivity, specificity, PPV, NPV, accuracy, LH+ and LH- of urinary iCOCI test for diagnosing CaOx urolithiasis were 95%, 86%, 68%, 98%, 88%, 6.80 and 0.06, respectively. Conclusion, we clinically validated that an innovative non-invasive urinary iCOCI test was highly accurate to diagnose urolithiasis, especially CaOx stone. With its high sensitivity and NPV, urinary iCOCI test is clinically intended to use as a screening test for CaOx urolithiasis. LH- of 0.06 indicates that negative result of urinary iCOCI test is highly accurate to rule out the CaOx stone formation. It is noted that urinary iCOCI level is expressed as arbitrary unit, and it is not directly related to the actual physiological level of urinary oxalate.