Pancreatic cancer is an aggressive disease in which malignant cells form in the tissues of the pancreas, a long and flat gland located behind the stomach that helps with digestion and blood sugar regulation.
Because pancreatic cancer is difficult to detect early, it is associated with a low survival rate, accounting for just over 3% of all new cancer cases in the U.S., but leading to nearly 8% of all cancer deaths, according to the National Cancer Institute.
Through a pre-clinical study conducted in his former role at Moffitt Cancer Center and published in Clinical Cancer Research, Said Sebti, Ph.D., associate director for basic research at VCU Massey Cancer Center, identified a novel drug that effectively thwarts pancreatic tumors that are addicted to the cancer-causing mutant KRAS gene. Sebti recently met with clinical colleagues at Massey to discuss evaluating the drug in clinical trials in patients whose pancreatic tumors harbor mutant KRAS.
“We discovered a link between hyperactivation of the CDK protein and mutant KRAS addiction, and we exploited this link preclinically to counter mutant KRAS-driven pancreatic cancer, warranting clinical investigation in patients afflicted with this deadly disease,” said Sebti, who is also the Lacy Family Chair in Cancer Research at Massey and a professor of pharmacology and toxicology at the VCU School of Medicine.
“Our findings are highly significant as they revealed a new avenue to combat an aggressive form of pancreatic cancer with very poor prognosis due mainly to its resistance to conventional therapies.”
KRAS is mutated in 90 percent of pancreatic cancers. Previous research from the Sebti lab and other labs has demonstrated that some tumors that harbor mutant KRAS are actually addicted to the mutant gene, meaning they cannot survive or grow without it.
Sebti set out to discover if there is a drug that can specifically kill tumors that are addicted to mutant KRAS.
Sebti and collaborators used three scientific approaches to try and answer this question.
First, they mapped out the blueprint of pancreatic cancer cells through global phosphoproteomics, which gave them a snapshot of how the addicted and non-addicted tumors differ at the phosphoprotein level.
They found two proteins – CDK1 and CDK2 – which were indicative of which cells were addicted to mutant KRAS.
Additionally, they analyzed a comprehensive database from the Broad Institute of MIT and Harvard that contains genome-wide CRISPR gRNA screening datasets.
Lastly, they evaluated the ability of a library of 294 FDA drugs to selectively kill mutant KRAS-addicted cancer cells over non-KRAS-addicted cancer cells in the lab and determined the most effective drug in preclinical experiments was AT7519, an inhibitor of CDK1, CDK2, CDK7 and CDK9.
“Using three entirely different approaches, the same conclusion presented itself clearly to us: pancreatic cancer patients whose tumors are addicted to mutant KRAS could benefit greatly from treatment with the CDK inhibitor AT7519,” Sebti said.
To further validate these findings in fresh patient-derived tumors from pancreatic cancer patients, Sebti collaborated on this study with Jose Trevino, M.D., surgeon-in-chief and the Walter Lawrence, Jr., Distinguished Professorship in Oncology at Massey who was at the University of Florida at the time. They found that AT7519 suppressed the growth of xenograft cells from five mutant KRAS pancreatic cancer patients who relapsed on chemotherapy and/or radiation therapies.
AT7519 has previously been tested unsuccessfully in a number of clinical trials, but none of the trials targeted pancreatic cancer.
“If our findings are correct and translate in humans, then we should be able to see a positive response in pancreatic cancer patients whose tumors are addicted to mutant KRAS,” Sebti said.
The study authors believe that, in addition to pancreatic cancer, these findings may also have clinical implications for colorectal and non-small cell lung cancer patients where mutations in KRAS are prevalent.
Pancreatic ductal adenocarcinoma (PDAC) is the deadliest form of pancreatic cancer and accounts for mortality among a majority of pancreatic cancer patients (Siegel et al., 2016). It is estimated that in 2019, 3.2% of all new cases and 7.5% of all cancer-related deaths will be due to pancreatic cancer (https://seer.cancer.gov/statfacts/html/pancreas.html). Limited therapeutic options as a result of late stage diagnosis have contributed to the poor clinical outcome, with only ∼7% of PDAC patients reaching the 5-year survival mark (Siegel et al., 2016).
A common hallmark of cancer is evasion of cell death, which contributes to the persistence of pancreatic cancer and its resistance to chemotherapy (Westphal and Kalthoff, 2003). Elevated expression of antiapoptotic protein levels (Bcl-xL and Mcl-1) observed in PDAC suggests that the apoptotic pathway is deregulated (Campani et al., 2001; Evans et al., 2001) and targeting them is a viable therapeutic strategy for PDAC (Takahashi et al., 2013; Abulwerdi et al., 2014).
Recent studies demonstrated that concurrent inactivation of Bcl-xL and Mcl-1 resulted in robust induction of apoptosis (Lopez et al., 2010; Rajule et al., 2012; O’Neill et al., 2016). As a result, Bcl-xL and Mcl-1 are considered attractive therapeutic targets for cancer therapy (Tse et al., 2008; Touzeau et al., 2011; Leverson et al., 2015; Contreras et al., 2018).
Abbott Laboratories (ABT) successfully developed direct inhibitors of Bcl-xL/Bcl-2/Bcl-w (the first drugs to target Bcl-2 family proteins): 4-{4-[(4′-chloro-2-biphenylyl)methyl]-1-piperazinyl}-N-[(4-{[(2R)-4-(dimethylamino)-1-(phenylsulfanyl)-2-butanyl]amino}-3-nitrophenyl) sulfonyl]benzamide (ABT-737); 4-(4-{[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-[(4-{[(2R)-4-(4-morpholinyl)-1-(phenylsulfanyl)-2-butanyl]amino}-3-[(trifluoromethyl)sulfonyl]phenyl)sulfonyl] benzamide [(ABT-263), navitoclax]; and 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide (Tse et al., 2008; Touzeau et al., 2011).
However, resistance to Bcl-xL/Bcl-2/Bcl-w inhibition has been attributed to compensatory activity by Mcl-1 (Konopleva et al., 2006; Mazumder et al., 2012). Consequently, Mcl-1 inactivation sensitizes cancer cells to Bcl-xL/Bcl-2/Bcl-w inhibitors (Peddaboina et al., 2012; Mohammad et al., 2017). While the development of direct Mcl-1 inhibitors is currently in preclinical and clinical development (AMG-176 [Spiro[5,7-etheno-1H,11H-cyclobut[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-2(3H),1′(2′H)- naphthalen]-8(9H)-one, 6′-chloro-3′,4′,12,13,16,16a,17,18,18a,19-decahydro-16-methoxy-11,12-dimethyl- , 10,10-dioxide, (1′S,11R,12S,14E,16S,16aR,18aR)-], AZD5991 [(Z)-16-chloro-11,21,25,61-tetramethyl-11H,21H,61H-10-oxa-4,8-dithia-1(7,3)-indola-2(4,3),6(3,5)-dipyrazola-9(3,1)-naphthalenacyclotridecaphane-12-carboxylic acid], and S63845 [(R)-2-((5-(3-chloro-2-methyl-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-((1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl)methoxy)phenyl)propanoic acid]) (Kotschy et al., 2016; Letai, 2016; Merino et al., 2017) targeting Mcl-1 indirectly is an alternate approach.
Several members of the cyclin-dependent kinases (CDKs) regulate the stability and expression of Mcl-1. Therefore, an alternate strategy to target Mcl-1 would be through modulation of members of the CDK family (MacCallum et al., 2005; Lowman et al., 2010; Choudhary et al., 2015b). Specifically, CDK5 regulates Mcl-1 function through phosphorylation of the endogenous Mcl-1 antagonist, NOXA (Lowman et al., 2010).
The kinase activity of CDK5 is regulated by p35, which has a myristoylation site at the N-terminus. In pancreatic tumors and cell lines, we previously showed the presence of elevated p25 levels, which is a cleaved product of p35 (Eggers et al., 2011). The cleavage of p35 to p25 leads to two changes: 1) mislocalization of CDK5/p25 to the cytoplasm due to loss of myristoylated anchoring by p35, and 2) increased half-life of CDK5/p25 (Patrick et al., 1999). These changes in CDK5 function/regulation along with the high homology observed between CDK2 and CDK5 indicate that in pancreatic cancer, CDK2 phosphorylation sites on Mcl-1 could now be targeted by CDK5/p25 (Kobayashi et al., 2007). Together, these studies suggest that a CDK5 inhibitor will disable Mcl-1 function and can be used in combination with Bcl-2/Bcl-xL/Bcl-w inhibitors as a promising therapeutic strategy for pancreatic cancer.
Small molecules containing the aminopyrazole core have shown promise as CDK inhibitors (Pevarello et al., 2004; Robb et al., 2017). Recent work from our laboratory characterized an aminopyrazole analog (CP-668863 [N-(3-cyclobutyl-1H-pyrazol-5-yl)-2-(naphthalen-2-yl)acetamide]) that inhibited tumor growth in a xenograft model of colorectal cancer (Robb et al., 2018). Using CP-668863 as a guide, we synthesized a focused library of aminopyrazole analogs and identified a potent CDK2/5 inhibitor, 2-([1,1′-biphenyl]-4-yl)-N-(5-cyclobutyl-1H-pyrazol-3-yl)acetamide (analog 24).
We found that on an average (in five pancreatic cancer cell lines) analog 24 was ∼2-fold more potent than AT7519 [4-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide] and ∼45-fold more potent than roscovitine [(2R)-2-[[6-(benzylamino)-9-propan-2-ylpurin-2-yl]amino]butan-1-ol] (Rana et al., 2018).
Here, we show that analog 24 selectively inhibits the kinase activity of CDK5 over CDK2 in cancer cells and results in reduced Mcl-1 levels. We demonstrate selective perturbation of Mcl-1 function over Bcl-2/Bcl-xL/Bcl-w by analog 24 in cells using a chemical genetic study. Moreover, in pancreatic cancer cell lines, CDK5 knockdown or analog 24 treatment synergistically induced apoptosis when combined with the Bcl-2 inhibitor navitoclax.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726458/
More information: Aslamuzzaman Kazi et al, Global Phosphoproteomics Reveal CDK Suppression as a Vulnerability to KRas Addiction in Pancreatic Cancer, Clinical Cancer Research (2021). DOI: 10.1158/1078-0432.CCR-20-4781
