ASPP2 is a tumor suppressor that regulates key proteins such as p53, Bcl-2 and NFkB to orchestrate pivotal cancer-related pathways. ASPP2 contains a highly conserved Ank-SH3 domain at its C-terminus, which mediates these interactions. Importantly, we discovered a so far unknown, oncogenic isoform, named ASPP2k, with high prevalence in cancer. This dominant-negative mutant has lost the crucial C-terminus and inhibits ASPP2 WT function, resulting in loss of tumor suppressing capacities. ASPP2k was first detected in acute leukemias, but screening of multiple tumor entities revealed frequent expression of ASPP2k in cancer, going along with a more aggressive tumorbiology and resistance to therapy.

> 500 tumors including AML, glioma, sarcoma, breast, colon and lung cancer, were assessed for ASPP2k expression. ASPP2k was stably suppressed or overexpressed in cell lines from these entities and in patient tissue. Induction of apoptosis, cellular proliferation, migration, invasion, colony formation, telomer length, angiogenesis and related pathways were assessed in dependency of ASPP2k. 6 independent, ASPP2k-modified mouse tumor models were established and tumor engraftment, progression and metastasis assessed in vivo.

Representative data, exemplary TNBC: Expression of ASPP2κ was confirmed in virtually all patients. ASPP2κ expressing TNBC cell models displayed impaired induction of apoptosis (avg. IC50s -30 %), higher proliferation (avg.+25%), migration (avg.+65%) and invasion (avg.+70%) rates. Knock down (KD) of ASPP2k sensitized cells towards chemotherapy (Doxorubicin avg.+35%) and/or γ-irradiation (avg.+35%) by restoring important p-p53 sites (S15: 2,5x/S46: 2x) and cell cycle regulators (p21: 2x, pRAD: 3x). EMT and angiogenesis were strongly inhibited in ASPP2k KD models, evidenced by downregulation of pro-angiogenic and pro-metastatic players (e.g. VEGF 90%, IL8 60%, Slug 70%, Snail 65%, n-Cadherin 40%). Xenotransplant ASPP2κ KD tumor mouse models confirm attenuated tumor engraftment, growth and metastasis (p=0.001), resulting in significantly prolonged overall survival (p=0.0001), Fig.1.

We demonstrate that ASPP2κ is highly expressed in cancer and intimately involved in tumorigenesis and drug resistance, promoting all classical hallmarks of cancer. Importantly, ASPP2k is targetable in vivo and we will provide data evaluating ASPP2κ as a novel target for therapy.