High iASPP (PPP1R13L) expression is an independent predictor of adverse clinical outcome in acute myeloid leukemia (AML)

Abstract

Apoptosis-stimulating proteins of p53 (ASPPs) are a family of proteins that modulate key tumor suppressor pathways via direct interaction with p53. Deregulation of these proteins promotes cancer development and impairs sensitivity to systemic (chemo)therapy and radiation. In this study, we describe that the inhibitor of ASPP (iASPP) is frequently highly expressed in acute myeloid leukemia (AML) and that overexpression correlates with a poor clinical outcome. Four independent patient cohorts comprising about 1500 patient samples were analysed and consistently confirm an association of high iASPP expression with unfavourable clinical characteristics and shorter survival. Notably, the predictive role of iASPP is independent of, and adds information to, the European LeukemiaNET (ELN) risk classification. iASPP-interference cell models were developed to investigate the underlying functional aspects of iASPP in AML biology. Attenuation of iASPP expression resulted in reduced proliferation rates of leukemic blasts and rendered cells more susceptible towards induction of apoptosis in response to cytotoxic therapy. In line, independent NSG xenograft mouse experiments demonstrate that attenuation of iASPP results in a significant delay of disease onset and tumor burden and this translates to longer overall survival of mice. In conclusion, deregulation of iASPP has direct functional consequences in AML. Determination of iASPP expression levels provides valuable additional information as a predictive marker in AML and may guide treatment decisions.

Fig. 1. iASPP expression in AML.

A Relative iASPP mRNA expression levels of a large RNAseq dataset including n = 296 pts with AML compared to physiologic whole blood samples, n = 670. B Relative iASPP mRNA expression levels of 43 AML patients compared to samples from healthy bone marrow (BM) donors (n = 11). GAPDH served as a housekeeping gene. C Immunohistochemistry stains (40x) of iASPP in bone marrow of a healthy donor and a patient with AML. D Relative iASPP protein expression levels in 73 patients with newly diagnosed AML compared to mononuclear cells from 31 healthy bone marrow (BM) donors as detected by immunostaining in a flow cytometer approach. Isotype IgG controls served as basal levels. E iASPP expression of leukemia blasts derived from bone marrow (n = 26) or peripheral blood (n = 55). F−I iASPP expression according to (F) complete remission (CR), n = 10, vs. pts. failing CR, n = 22, p = 0.272, (G) AML qualifiers (de novo AML, n = 42, vs. AML progressing from MDS or MDS/MPN and therapy related AML according to the ICC 2022 classification [23] (ref. to as secondary AML), n = 24, (H) newly diagnosed AML vs. R/R AML (n = 10) and (I) ELN 2017 risk stratification (healthy bm donors (n = 31), favorable risk (n = 15), intermediate risk (n = 19), adverse risk (n = 23)). ns (not significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Mann-Whitney-U-Test).

Fig. 2. iASPP and survival in AML.

A, C, E, G OS and B, D, F, H EFS according to iASPP expression and risk category according to karyotype in a transcriptomic dataset (GSE6891).

Fig. 3. iASPP and survival in a patient cohort treated in the HOVON102 trial (n = 274).

A OS and B EFS according to iASPP expression level. C–E Contingency analysis: proportion of event-free survival at 3 (C), 6 (D) and 12 (E) months after achieving remission. F OS, G EFS hazard ratios and I median OS data according to iASPP expression levels and ELN 2017 risk score (favorable/intermediate/adverse risk +/- iASPP_low or iASPP_high).

Fig. 4. Functional analysis of iASPP in a MOLM14 interference cell line model.

A, B Transduction efficiency assessed by (A) mRNA assessed by RT-qPCR and (B) protein levels assessed by flow cytometry. C iASPP-interference results in reduced metabolic activity as assessed by XTT, D corresponding to an attenuated proliferation rate with a prolonged cellular doubling time (16 h for iASPP KD vs. 13 h for iASPP EV). E, F, G Induction of apoptosis in response to daunorubicin, cytarabine or FLT3/multikinase-inhibitor sunitinib. H, I, J Box plot analysis to determine IC₅₀ concentrations compared to empty vector (EV) control strains. Technical triplicates minimum for all assays. *p ≤ 0.05, **p ≤ 0.01, ≤ ***p ≤ 0.001, ****p ≤ 0.0001 (t-test).

Fig. 5. Apoptosis signaling of MOLM14 iASPP-interferenced vs. EV cell strains in response to daunorubicin.

A Heat map of a proteome profiler human apoptosis array covering 35 proteins involved in apoptosis control. B Upregulation of proteins involved in induction of apoptosis and cell cycle control in the iASPP knock down (KD) vs EV cell strains.

Fig. 6. Xenotransplant mouse model according to iASSP expression.

A Transduction efficiency assessed by mRNA. B In vivo visualization of luciferase activity in MOLM14_iASPP KD_Luc+ vs. MOLM14_EV_Luc+ xenotransplanted mice on day 26 post transplantationem. 3 representative mice shown. C Luciferase activity over time. D Survival rates over time (n = 4 EV / 4 KD). E Whole femur of a representative MOLM14 EV control mouse, stained with DAPI, tdTomato, Endomucin, aSMA and CollagenIV, 10x magnification. F, G Zoomed-in images of BM regions that were densely infiltrated by MOLM14 cells. (H) MOLM14-iASPP KD Luc+ mouse femur with a single MOLM14 infiltration zone under the growth plate of the distal epiphysis and (I, J) zoomed-in image of the proximal epiphysis. **p ≤ 0.01 (Log-Rank Mantel Cox test), ***p ≤ 0.001 (unpaired t-test).