Abstract Background ASPP2 is a critical p53 activator initiating apoptotic cascades in the presence of cell stress. ASPP2k is a novel, dominant-negative, oncogenic splicing isoform of the tumor suppressor ASPP2, discovered in the context of a mutational screening of AML/ALL patient biopsies (expressed in >40% of the samples) (Schittenhelm MM et atl, EBioMed 2019). ASPP2k results from an aberrant exon 17 skipping: a premature STOP codon at the new exon16-exon18 junction site leads to the translation of a truncated protein. Indeed, ASPP2k lacks the ASPP2 C-terminus, containing the important p53 binding sites. Thus, ASPP2k impairs p53-dependent apoptosis activation, resulting in accelerated proliferation and attenuated chemotherapy-induced apoptosis in cancer cells. Results ASPP2k silencing (KD) and overexpression (KI) functional consequences were evaluated in vitro and in vivo. ASPP2k was silenced in MOLM-14 cells and primary AML blasts. We found proliferation to be attenuated in ASPP2k KD cells compared to EV cells in both models (p<0.0001). Silencing of ASPP2k rendered cells more susceptible towards standard chemotherapy compared to controls (+30% Daunorubicin 10 nM, +20% Venetoclax 15 nM, p<0.01, Annexin/PI). Both cell models were subsequently injected intravenously into NSG mice: ASPP2k KD lead to a less aggressive disease development, resulting in a longer lifespan (30 and 42 days for EV and KD, respectively). Significant splenomegaly was noted for all control mice, while spleens from mice injected with ASPP2k KD cells were on average half in size and weight, arguing for a less aggressive biology of KD leukemia cells. Vice versa, ASPP2k up-regulation resulted in opposite results. IL-3 dependent Ba/F3 cells are a standard model to evaluate the oncogenic capacities of potential tumor drivers. Upon ASPP2k KI, Ba/F3 cells lost their IL-3 dependency. In line, overexpression of ASPP2k resulted in accelerated proliferation (+9.32 and +127.75 fold-change at 96h for EV and KI cells respectively, compared to 24h). When injected into NSG mice, ASPP2k KI Luc+ cells spread rapidly and invaded spleen, liver and brain, while no disease dissemination was noticeable control mice. Data from regular blood counts confirmed this notion. Animals injected with ASPP2k KI cells showed a rapid disease progression, while EV mice showed no signs of the illness. Further an increase of +122.5% in spleen weight was assessed in mice injected with ASPP2k KI cells compared to control animals at termination day, further underlining oncogenic potential of ASPP2k. Conclusions In summary, we here show that ASPP2κ directly contributes to resistance towards therapy in AML as well as aggressiveness of disease in vivo in all the investigated models, arguing for an oncogenic potential of the variant. Future studies evaluating ASPP2κ as a prognostic/predictive model and potential target for therapy are warranted. Citation Format: Alessia Ruiba, Marcus Schittenhelm, Anna Lena Ahrens, Vanessa Aellig, Kerstin Kampa-Schittenhelm. Expression of oncogenic ASSP2k in acute myeloid leukemia directly contributes to resistance to therapy and aggressiveness of the disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5992.