Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.

The DNAJ-PKAc fusion kinase is a defining feature of fibrolamellar carcinoma (FLC). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. By combining proximity proteomics, biochemical analyses, and live-cell photoactivation microscopy, we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates, including proteins involved in translation and the anti-apoptotic factor Bcl-2-associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Tissue samples from patients with FLC exhibit increased levels of BAG2 in primary and metastatic tumors. Furthermore, drug studies implicate the DNAJ-PKAc/Hsp70/BAG2 axis in potentiating chemotherapeutic resistance. We find that the Bcl-2 inhibitor navitoclax enhances sensitivity to etoposide-induced apoptosis in cells expressing DNAJ-PKAc. Thus, our work indicates BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.

Oncogenic PKA signaling increases c-MYC protein expression through multiple targetable mechanisms.

Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.

Hybrid Capture-Based Tumor Sequencing and Copy Number Analysis to Confirm Origin of Metachronous Metastases in BRCA1-Mutant Cholangiocarcinoma Harboring a Novel YWHAZ-BRAF Fusion.

Biliary tract cancers such as cholangiocarcinoma represent a heterogeneous group of cancers that can be difficult to diagnose. Recent comprehensive genomic analyses in large cholangiocarcinoma cohorts have defined important molecular subgroups within cholangiocarcinoma that may relate to anatomic location and etiology [1], [2], [3], [4] and may predict responsiveness to targeted therapies in development [5], [6], [7]. These emerging data highlight the potential for tumor genomics to inform diagnosis and treatment options in this challenging tumor type. We report the case of a patient with a germline BRCA1 mutation who presented with a cholangiocarcinoma driven by the novel YWHAZ-BRAF fusion. Hybrid capture-based DNA sequencing and copy number analysis performed as part of clinical care demonstrated that two later-occurring tumors were clonally derived from the primary cholangiocarcinoma rather than distinct new primaries, revealing an unusual pattern of late metachronous metastasis. We discuss the clinical significance of these genetic alterations and their relevance to therapeutic strategies. KEY POINTS: Hybrid capture-based next-generation DNA sequencing assays can provide diagnostic clarity in patients with unusual patterns of metastasis and recurrence in which the pathologic diagnosis is ambiguous.To our knowledge, this is the first reported case of a YWHAZ-BRAF fusion in pancreaticobiliary cancer, and a very rare case of cholangiocarcinoma in the setting of a germline BRCA1 mutation.The patient's BRCA1 mutation and YWHAZ-BRAF fusion constitute potential targets for future therapy.