Phenotype-Based Screens with Conformation-Specific Inhibitors Reveal p38 Gamma and Delta as Targets for HCC Polypharmacology

Abstract

The approved kinase inhibitors for hepatocellular carcinoma (HCC) are not matched to specific mutations within tumors. This has presented a daunting challenge; without a clear target or mechanism, no straightforward path has existed to guide the development of improved therapies for HCC. Here, we combine phenotypic screens with a class of conformation-specific kinase inhibitors termed type II to identify a multikinase inhibitor, AD80, with antitumoral activity across a variety of HCC preclinical models, including mouse xenografts. Mass spectrometry profiling found a number of kinases as putative targets for AD80, including several receptor and cytoplasmic protein kinases. Among these, we found p38 gamma and delta as direct targets of AD80. Notably, a closely related analog of AD80 lacking p38d/g activity, but retaining several other off-target kinases, lost significant activity in several HCC models. Moreover, forced and sustained MKK6!p38!ATF2 signaling led to a significant reduction of AD80 activity within HCC cell lines. Together with HCC survival data in The Cancer Genome Atlas and RNA-seq analysis, we suggest p38 delta and gamma as therapeutic targets in HCC and an "AD80 inhibition signature" as identifying those patients with best clinical outcomes.

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“…Our findings are in agreement with Liu et al showing a durable suppression of S6K1-induced signaling by AD80 in PTEN-deficient tumors [20]. Similarly, Yu et al, identified an AD80 as a lead compound profoundly inhibiting oncogenic signaling in hepatocellular carcinoma [45].…”

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“…On the other hand, compound 124 adopted a DFG-in binding mode and showed lower inhibitory activity against p38δ. However, all the three designed compounds also inhibited the activity of p38α, which shows that the changes made in their structures were not sufficient to interfere with the binding to other p38 DFG-out [47] WFYH compound SU-005 was synthesized changing the octanamide of SU-002 by an oct-2-enamide attached to CQT structure (Table 1) [86]. SU-005 specifically decreased p38δ and p38γ activity both in vitro and in cells lines and did not inhibit p38δ with a Met107Thr mutant, suggesting that this compound may be a specific inhibitor of p38δ and p38γ owing to the presence of the gatekeeper residue Met [86].…”

“…In contrast to its tumor suppressor roles, p38δ also has tumor promoter roles, as evidenced in several types of cancers, such as skin carcinoma [ 29 , 44 , 45 ], pleural malignant mesothelioma (PMM) [ 46 ], HCC [ 47 ], colitis-associated colorectal cancer (CAC) [ 33 ], head and HNSCC [ 28 , 30 ], CC [ 26 ], and breast cancer [ 27 ]. The potential tumor promoter roles of p38δ are summarized in Figure 1 .…”

“…Another indication for a specific role of p38δ in promoting cancer progression has recently been demonstrated in HCC. This study identified a multikinase inhibitor, compound AD80, with p38γ and p38δ as its targets and antitumoral activity across a variety of HCC in vivo and in vitro models [ 47 ]. Moreover, growth inhibition assays and long-term clonogenic assays on HCC cell lines revealed that AD80 was more potent in these cells than Sorafenib, a compound commonly used in HCC therapy [ 47 ].…”

“…This study identified a multikinase inhibitor, compound AD80, with p38γ and p38δ as its targets and antitumoral activity across a variety of HCC in vivo and in vitro models [ 47 ]. Moreover, growth inhibition assays and long-term clonogenic assays on HCC cell lines revealed that AD80 was more potent in these cells than Sorafenib, a compound commonly used in HCC therapy [ 47 ]. The potential pro-oncogenic role of p38γ and p38δ on aggressive HCC was confirmed by analysis of The Cancer Genome Atlas (TCGA) liver cancer dataset.…”