A novel treatment strategy utilizing panobinostat for high-risk and treatment-refractory hepatoblastoma.

BACKGROUND & AIMS: Patients with metastatic, treatment-refractory, and relapsed hepatoblastoma (HB) have survival rates of less than 50% due to limited treatment options. To develop new therapeutic strategies for these patients, our laboratory has developed a preclinical testing pipeline. Given that histone deacetylase (HDAC) inhibition has been proposed for HB, we hypothesized that we could find an effective combination treatment strategy utilizing HDAC inhibition. METHODS: RNA sequencing, microarray, NanoString, and immunohistochemistry data of patient HB samples were analyzed for HDAC class expression. Patient-derived spheroids (PDSp) were used to screen combination chemotherapy with an HDAC inhibitor, panobinostat. Patient-derived xenograft (PDX) mouse models were developed and treated with the combination therapy that showed the highest efficacy in the PDSp drug screen. RESULTS: HDAC RNA and protein expression were elevated in HB tumors compared to normal livers. Panobinostat (IC50 of 0.013-0.059 µM) showed strong in vitro effects and was associated with lower cell viability than other HDAC inhibitors. PDSp demonstrated the highest level of cell death with combination treatment of vincristine/irinotecan/panobinostat (VIP). All four models responded to VIP therapy with a decrease in tumor size compared to placebo. After 6 weeks of treatment, two models demonstrated necrotic cell death, with lower Ki67 expression, decreased serum alpha fetoprotein and reduced tumor burden compared to paired VI- and placebo-treated groups. CONCLUSIONS: Utilizing a preclinical HB pipeline, we demonstrate that panobinostat in combination with VI chemotherapy can induce an effective tumor response in models developed from patients with high-risk, relapsed, and treatment-refractory HB. IMPACT AND IMPLICATIONS: Patients with treatment-refractory hepatoblastoma have limited treatment options with survival rates of less than 50%. Our manuscript demonstrates that combination therapy with vincristine, irinotecan, and panobinostat reduces the size of high-risk, relapsed, and treatment-refractory tumors. With this work we provide preclinical evidence to support utilizing this combination therapy as an arm in future clinical trials.

CD203c is expressed by human fetal hepatoblasts and distinguishes subsets of hepatoblastoma.

Background & Aims: Hepatocytic cells found during prenatal development have unique features compared to their adult counterparts, and are believed to be the precursors of pediatric hepatoblastoma. The cell-surface phenotype of hepatoblasts and hepatoblastoma cell lines was evaluated to discover new markers of these cells and gain insight into the development of hepatocytic cells and the phenotypes and origins of hepatoblastoma. Methods: Human midgestation livers and four pediatric hepatoblastoma cell lines were screened using flow cytometry. Expression of over 300 antigens was evaluated on hepatoblasts defined by their expression of CD326 (EpCAM) and CD14. Also analyzed were hematopoietic cells, expressing CD45, and liver sinusoidal-endothelial cells (LSECs), expressing CD14 but lacking CD45 expression. Select antigens were further examined by fluorescence immunomicroscopy of fetal liver sections. Antigen expression was also confirmed on cultured cells by both methods. Gene expression analysis by liver cells, 6 hepatoblastoma cell lines, and hepatoblastoma cells was performed. Immunohistochemistry was used to evaluate CD203c, CD326, and cytokeratin-19 expression on three hepatoblastoma tumors. Results: Antibody screening identified many cell surface markers commonly or divergently expressed by hematopoietic cells, LSECs, and hepatoblasts. Thirteen novel markers expressed on fetal hepatoblasts were identified including ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP-3/CD203c), which was found to be expressed by hepatoblasts with widespread expression in the parenchyma of the fetal liver. In culture CD203c+CD326++ cells resembled hepatocytic cells with coexpression of albumin and cytokeratin-19 confirming a hepatoblast phenotype. CD203c expression declined rapidly in culture whereas the loss of CD326 was not as pronounced. CD203c and CD326 were co-expressed on a subset of hepatoblastoma cell lines and hepatoblastomas with an embryonal pattern. Conclusions: CD203c is expressed on hepatoblasts and may play a role in purinergic signaling in the developing liver. Hepatoblastoma cell lines were found to consist of two broad phenotypes consisting of a cholangiocyte-like phenotype that expressed CD203c and CD326 and a hepatocyte-like phenotype with diminished expression of these markers. CD203c was expressed by some hepatoblastoma tumors and may represent a marker of a less differentiated embryonal component.

Design considerations of an IL13Rα2 antibody-drug conjugate for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG), a rare pediatric brain tumor, afflicts approximately 350 new patients each year in the United States. DIPG is noted for its lethality, as fewer than 1% of patients survive to five years. Multiple clinical trials involving chemotherapy, radiotherapy, and/or targeted therapy have all failed to improve clinical outcomes. Recently, high-throughput sequencing of a cohort of DIPG samples identified potential therapeutic targets, including interleukin 13 receptor subunit alpha 2 (IL13Rα2) which was expressed in multiple tumor samples and comparably absent in normal brain tissue, identifying IL13Rα2 as a potential therapeutic target in DIPG. In this work, we investigated the role of IL13Rα2 signaling in progression and invasion of DIPG and viability of IL13Rα2 as a therapeutic target through the use of immunoconjugate agents. We discovered that IL13Rα2 stimulation via canonical ligands demonstrates minimal impact on both the cellular proliferation and cellular invasion of DIPG cells, suggesting IL13Rα2 signaling is non-essential for DIPG progression in vitro. However, exposure to an anti-IL13Rα2 antibody-drug conjugate demonstrated potent pharmacological response in DIPG cell models both in vitro and ex ovo in a manner strongly associated with IL13Rα2 expression, supporting the potential use of targeting IL13Rα2 as a DIPG therapy. However, the tested ADC was effective in most but not all cell models, thus selection of the optimal payload will be essential for clinical translation of an anti-IL13Rα2 ADC for DIPG.

Volasertib preclinical activity in high-risk hepatoblastoma.

Relapsed and metastatic hepatoblastoma represents an unmet clinical need with limited chemotherapy treatment options. In a chemical screen, we identified volasertib as an agent with in vitro activity, inhibiting hepatoblastoma cell growth while sparing normal hepatocytes. Volasertib targets PLK1 and prevents the progression of mitosis, resulting in eventual cell death. PLK1 is overexpressed in hepatoblastoma biopsies relative to normal liver tissue. As a potential therapeutic strategy, we tested the combination of volasertib and the relapse-related hepatoblastoma chemotherapeutic irinotecan. We found both in vitro and in vivo efficacy of this combination, which may merit further preclinical investigation and exploration for a clinical trial concept.

Induction of Mycobacterium Tuberculosis Lipid-Specific T Cell Responses by Pulmonary Delivery of Mycolic Acid-Loaded Polymeric Micellar Nanocarriers.

Mycolic acid (MA), a major lipid component of Mycobacterium tuberculosis (Mtb) cell wall, can be presented by the non-polymorphic antigen presenting molecule CD1b to T cells isolated from Mtb-infected individuals. These MA-specific CD1b-restricted T cells are cytotoxic, produce Th1 cytokines, and form memory populations, suggesting that MA can be explored as a potential subunit vaccine candidate for TB. However, the controlled elicitation of MA-specific T cell responses has been challenging due to difficulties in the targeted delivery of lipid antigens and a lack of suitable animal models. In this study, we generated MA-loaded micellar nanocarriers (MA-Mc) comprised of self-assembled poly(ethylene glycol)-bl-poly(propylene sulfide; PEG-PPS) copolymers conjugated to an acid sensitive fluorophore to enhance intracellular delivery of MA to phagocytic immune cells. Using humanized CD1 transgenic (hCD1Tg) mice, we found these nanobiomaterials to be endocytosed by bone marrow-derived dendritic cells (DCs) and localized to lysosomal compartments. Additionally, MA-Mc demonstrated superior efficacy over free MA in activating MA-specific TCR transgenic (DN1) T cells in vitro. Following intranasal immunization, MA-Mc were primarily taken up by alveolar macrophages and DCs in the lung and induced activation and proliferation of adoptively transferred DN1 T cells. Furthermore, intranasal immunization with MA-Mc induced MA-specific T cell responses in the lungs of hCD1Tg mice. Collectively, our data demonstrates that pulmonary delivery of MA via PEG-PPS micelles to DCs can elicit potent CD1b-restricted T cell responses both in vitro and in vivo and MA-Mc could be explored as subunit vaccines against Mtb infection.