(P 58) COMBINATORY THERAPIES UTILIZING INHIBITION OF CARBONIC ANHYDRASE IX TO POTENTIATE RESPONSE IN RELAPSED/REFRACTORY OSTEOSARCOMA

Objective: Osteosarcoma (OS) is the most prevalent bone tumor, primarily affecting children, adolescents, and young adults. Current treatments, including chemotherapy and surgical resection, have remained largely unchanged and are ineffective for metastatic OS, highlighting the need for novel therapies. Hypoxic niches within tumors contribute to therapeutic resistance. Hypoxia drives OS cells to make more lactic acid which is effluxed via the enzyme Carbonic anhydrase IX (CAIX), rendering the surrounding tumor microenvironment acidic and altering the immune cells. CAIX inhibition alone has not been proven to be an effective strategy in treating cancer for which the exploration of an effective combination with an FDA/EMA approved anti-cancer compounds needs to be identified that could be translated into clinical studies.

Methods: Drug sensitivity testing was performed utilizing a FDA/EMA approved library including 202 anti-cancer compounds, across three cell lines: CRISPR-Cas9 CAIX knock-out, short hairpin (sh) RNA CAIX knock down and wild-type (WT) murine OS cell line K7M2. Individual drugs were dissolved in 100% DMSO and tested in duplicates starting at a maximal test concentration of 10μM then over a 20,000-fold concentration range to generate dose-response curves. Wells with assay buffer containing DMSO served as negative controls. 1000 exponentially growing cells were then seeded per well in 384-well micro-titer plates and incubated in the presence of compounds in a humidified environment at 37°C and 5% CO2. Cell viability was assessed after 72 hours of treatment, by measuring ATP levels via bioluminescence (CellTiter-Glo, Promega) and dose-response curves were generated for each compound.

Results: From the 202 compounds screened, 24 showed activity (HIT 35%) across all three cell lines. Drugs with maximal effect included purine analogs-- Clofarabine, cladribine; Histone deacetylase (HDAC) inhibitors- Panobinostat, Belinostat and Tyrosine Kinase Inhibitors- Ponatinib, Dasatinib. None of the compounds demonstrated selective activity against CAIX-inhibited cells compared to WT cells, except for Regorafenib, which showed significant potentiation of response in CAIX Knock out cells as compared to WT (4.27± 0.14 % viability vs 61.65 ± 28.95 %).

Conclusion: The screening identified 24 compounds with broad activity across CAIX knock-out/down and WT OS cell lines, that require further validation and exploration under hypoxic conditions, which is ongoing. Regorafenib shows enhanced activity against CAIX-inhibited cells, therefore combining a CAIX inhibitor with regorafenib presents a potential therapeutic advance and should be studied in more detail.