DNAJB1-PRKACA peptide vaccine

Two doses of the DNAJB1-PRKACA fusion-based neoepitope vaccine Fusion-VAC-XS15 will be applied subcutaneously (s.c.) with a 4-week interval in combination with the anti-programmed cell death-ligand 1 (PD-L1) antibody atezolizumab starting at day 15 after the first vaccination. Clinical trial results will be published in peer-reviewed journals. EU CT Number: 2022-502869-17-01 and ClinicalTrials.gov Registry (NCT05937295).

Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity in vivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC.

Overall, we propose that LINC00473 could be a viable target for this devastating disease. Schematic was created with BioRender.com.

Neoplastic FALDs show some unusual molecular profiles compared with non-Fontan ones. The presence of the same alterations in non-lesional cardiac cirrhosis could contribute to the development of FALD.

Preclinical work from our laboratory and others has revealed that the DNAJB1-PRKACA fusion results in a metabolic rewiring of the tumor characterized by glutamine dependence. This study has been registered under NCT06027086 and is expected to begin enrollment in December 2023. Clinical trial information: NCT06027086.

Based on these encouraging results, we established a Phase I open-label, multicentric clinical trial to evaluate the immunogenicity along with safety and toxicity, as well as first signs of efficacy of the FusionVAC-22 based peptide vaccine, combined with the immune checkpoint inhibitor (ICI) atezolizumab, in 20 patients with locally advanced or metastatic FL-HCC or other malignant diseases that carry the DNAJB1-PRKACA fusion transcript. Furthermore, disease control rate, quality of life as well as overall and progression free survival will be assessed. Clinical trial information: NCT05937295.

Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.

The DNAJB1-PRKACA oncogenic gene fusion results in an active kinase enzyme, J-PKAcα, that has been identified as an attractive antitumor target for fibrolamellar hepatocellular carcinoma (FLHCC)...Human kinome profiling of 1 against a panel of 370 kinases revealed potent inhibition of select serine/threonine kinases in the CLK and PKG families with IC values in the range ∼11-90 nM. An efficient, four-step total synthesis of 1 has been accomplished, enabling further evaluation of aplithianines as biologically relevant kinase inhibitors.

We analyzed PRKACA/B fusion genes including ATP1B1-PRKACA, DNAJB1-PRKACA, and ATP1B1-PRKACB by reverse transcription PCR (RT-PCR); mRNA expression of fusion genes and non-rearranged PRKACA/B genes by quantitative RT-PCR; mutations in KRAS, BRAF, and GNAS by targeted sequencing or droplet digital PCR; and the expression of cAMP-dependent protein kinase catalytic subunit alpha (PRKACA) and beta (PRKACB), phosphorylated-CREB, and aberrations of p16, p53, SMAD4, STK11, and β-catenin by immunohistochemistry...In conclusion, PRKACA/B fusion genes are not only the characteristic drivers of IOPNs, but also play a crucial role in the development of subclonal oncocytic neoplasms. Moreover, oncocytic morphology is strongly associated with upregulation of PRKACA/B, which may provide clues for potential therapeutic options.

Results A young male with histologically confirmed FLC was treated with a 24mer DNAJB1-PRKACA synthetic long peptide vaccine with poly-ICLC in combination with ipilimumab and nivolumab on our clinical trial, NCT04248569. Our studies provide a framework to identify these responses in peripheral blood samples from treated patients and thereby determine correlates of response to therapy. Additionally, the defined fusion-specific TCRs show potential for translation to cellular therapies for FLC, highlighting multiple immunotherapeutic strategies that could benefit FLC patients.

P1, N=20, Recruiting, University Hospital Tuebingen | Not yet recruiting --> Recruiting

Most importantly, until now the second patient showed stable disease for 8 months after vaccination. Based on these promising data, we established a Phase I open label, multicentric clinical trial (EU-CT Number 2022-502869-17-00) evaluating the immunogenicity along with safety and toxicity, as well as first signs of efficacy of the FusionVAC-22 based peptide vaccine, combined with an ICI (anti-PD-L1), in 20 patients with locally advanced or metastatic FL-HCC or other malignant disease with proven presence of the DNAJB1-PRKACA fusion transcript.

FLC-Vac, consisting of a 24 amino acid peptide targeting the DNAJB1-PRKACA fusion with adjuvant poly-ICLC, is administered on weeks 0, 1, 2, 3, 6, 9 during the priming phase of the study and NIVO, 3 mg/kg, followed by IPI, 1 mg/kg, are administered every 3 weeks for 4 doses. Conclusions This first-in human study provides initial evidence of safety and clinical efficacy of a vaccine targeting the DNAJB1-PRKACA fusion plus immune checkpoint inhibitor therapy for FLC. T cell responses are consistent with neoantigen-specific immunity against the DNAJB1-PRKACA chimera.

We encountered a case of FL-HCC, a rare hepatic tumor in an adolescent patient, and evaluated the genetic background. Our findings could contribute to the elucidation of the mechanisms underlying carcinogenesis and progression in patients with FL-HCC and thereby contribute to the development of new therapeutic strategies in the future that may improve patient prognosis.

In regards to this tumour's genetic analysis, it is characterised by a 400 kb deletion on chromosome 19 leading to a functional DNAJB1-PRKACA chimeric transcript in addition to tetraploidy in 50% of cases...Although the prognosis for FLC is favourable as compared to other hepatic cancer subtypes such as intrahepatic cholangiocarcinoma, there is a high rate of recurrence ranging from 33% to 100% with a median recurrence-free survival of 20-48 months. As a result of this there is a low overall cure rate associated with this tumour type and much more research is required to gain an in-depth understanding of the molecular mechanisms occurring in order to provide more adequate treatment to patients who suffer from this condition.

P1, N=20, Not yet recruiting, University Hospital Tuebingen

Fusion protein DNAJB1-PRKACA is found in most of the cases...Recently, targeted therapy and immunotherapy have been studied which may provide survival advantage in the future. This review sought to compile data from clinical trials and case reports/series to outline the current state of FLC treatment.

In all analyses, DNAJB1-PRKACA organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.

P1, N=56, Recruiting, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins | N=12 --> 56 | Trial completion date: Mar 2024 --> Mar 2027 | Trial primary completion date: Mar 2024 --> Mar 2027

Fibrolamellar hepatocellular carcinoma (FLC) is a rare and often lethal form of liver cancer that primarily affects children and young adults. Systemic treatment of BALB/c mice bearing TIBx-FLC tumors with JHU-083, a glutamine antagonist, in combination with immune checkpoint inhibitor therapy enhanced survival as compared to vehicle or monotherapy. These data identify altered glutamine metabolism as a target in FLC, and may provide an explanation for immune suppression seen in the FLC tumor microenvironment.

Vaccine-induced DNAJB1-PRKACA-specific T cell responses persist over time and, in contrast to various previous treatments, are accompanied by durable relapse free survival of the patient for more than 21 months post vaccination. Our preclinical and clinical findings identify the DNAJB1-PRKACA protein as source for immunogenic neoepitopes and corresponding T cell receptors and provide efficacy in a single-patient study of T cell-based immunotherapy specifically targeting this oncogenic fusion.

Our results validate DNAJB1-PRKACA as the oncogene in FLC and demonstrate both a continued requirement for the oncogene for tumor growth as well as an oncogenic addiction that can be exploited for targeted therapies. We anticipate our approach will be useful for investigations of other fusion genes in pediatric cancers and spur development of precision therapies.

Recent advances in molecular studies of FLC have found a unique DNAJB1-PRKACA fusion transcript in most of the cases studied. The review aims to describe clinical characteristics, diagnostic methods, and therapeutic modalities for this rare tumor to raise awareness among clinicians and surgeons.

Furthermore, this compound showed anti-tumor activity in an FL-HCC patient-derived xenograft model expressing the DNAJB1-PRKACA fusion gene. Our data suggest that DS89002333 could be considered as a potential therapeutic agent for FL-HCC.

This study shows that, using standard techniques, FISH testing with a commercially available break apart probe targeting the PRKACA gene can be used as a surrogate for the DNAJB1-PRKACA fusion commonly found in FL-HCC. Also, the PRKACA gene rearrangement is not expressed in other hepatic neo-plasms/proliferations or extrahepatic neoplasms seen in children and young adults. Finally, FISH testing can be used as a diagnostic tool to confirm the diagnosis of FL-HCC, in the appropriate clinical setting.

This functional genomics study highlights a novel proliferative axis in FLC and provides a rich resource for further investigation of the molecular landscape of FLC. The results reveal that miR-10b-5p shapes gene expression and promotes cell proliferation in FLC. Future studies are necessary to identify how the loss of miR-455-3p contributes to FLC progression and how miR-10b-5p may coordinate with miR-455-3p to control tumor phenotypes.

In this Roadmap, we review advances since the seminal discovery in 2014 that nearly all FLC tumours express a signature oncogene (DNAJB1-PRKACA) encoding a fusion protein (DNAJ-PKAc) in which the J-domain of a heat shock protein 40 (HSP40) co-chaperone replaces an amino-terminal segment of the catalytic subunit of the cyclic AMP-dependent protein kinase (PKA). Important gains include increased understanding of oncogenic pathways driven by DNAJ-PKAc; identification of potential therapeutic targets; development of research models; elucidation of immune mechanisms with potential for the development of immunotherapies; and completion of the first multicentre clinical trials of targeted therapy for FLC. In each of these key areas we propose a Roadmap for future progress.

Ongoing experiments will test if cells expressing these TCRs can control growth of fusion-expressing tumors in vivo. Together, these studies have defined the first reported fusion-specific T cell response in an FLC patient, as well as fusion-specific TCRs that hold promise for development in adoptive T cell therapies.

FLC is driven by a fusion oncokinase, DNAJB1-PRKACA, that arises from a deletion fusing exon 1 of DNAJB1 and exons 2-10 of PRKACA, the catalytic subunit of protein kinase A. Expression of this chimeric oncokinase is capable of recapitulating the key histologic and transcriptomic features of FLC...Unfortunately, Navitoclax has an on-target and dose-limiting toxicity of thrombocytopenia, which limits its clinical application...In conclusion, TOPO1 and Bcl-xL prove to be promising targets of interest in FLC. Targeting both with DT2216 and irinotecan leads to tumor shrinkage in pre-clinical models and offer a potential therapeutic/pharmacological intervention for FLC.

DNAJB1-PRKACA-specific T cells persisted in peripheral blood and were accompanied by relapse free survival of the patient until now, more than one year post vaccination. These findings identified the DNAJB1-PRKACA fusion transcript as novel prime source for broadly applicable neoepitopes and corresponding TCRs and provide first evidence for their application in cancer immunotherapy of FL-HCC.

Collectively, these studies have defined the first reported fusion-specific T cell response in FLC, as well as fusion-specific TCRs that hold promise for use in adoptive T cell therapies. Our spatial transcriptomic analyses have also begun to illuminate the immune microenvironment in FLC and will inform future efforts to develop immunotherapies for this disease.

LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.

We also treated cells with PKA inhibitors that function by two different mechanisms (rpcAMPs and PKI) and examined phosphoproteome profiles, finding some substrates that persisted in the presence of inhibitors and revealing differences between WT and chimera. Overall, these results provide potential insights into J-PKA's oncogenic activity in a complex cellular system and may provide candidate targets for therapeutic follow-up.