PS1226 DEVELOPMENT OF INNOVATIVE PRECLINICAL IN VITRO AND IN VIVO TOOLS FOR AN EFFECTIVE THERAPEUTIC STRATEGY IN PEDIATRIC ACUTE MYELOID LEUKEMIA
Authors: G. Borella, V. Bisio, A. Da Ros, C. Tregnago, M. Benetton, E. Campodoni, S. Panseri, M. Sandri, M. Montesi, S. Cairo, F. Locatelli, M. Pigazzi
Journal: HemaSphere (2019)

Background: Diagnosis, treatment, minimal residual disease monitoring, and outcome of pediatric acute myeloid leukemia (AML) have made enormous progress during the past decade, although chemotherapy is still the pillar of pediatric treatment. Most of the emerged anti‐leukemic agents failed during experimentation, and one main limit in AML field is the inappropriateness of current pre‐clinical models used to study drug efficacy, reducing the advance of phase II and III clinical trials, especially for children. Aims: Set up and characterization of long term 3D‐AML cultures. Perform high throughput drug screening in vitro , and selection of best compounds to be then used in pre‐clinical AML‐PDX models. We would create a robust in vitro and in vivo pipeline to discover/reposition alternative treatments to improve AML children cure. Methods: The 3D structure is made up of engineered hydroxyapatite and collagen I to mimic endosteal bone marrow niche. We cultured mesenchymal stem cells derived from an AML patient (AML‐MSCs) together with its blasts. We studied 3D cultures also by using MSCs derived from healthy bone marrow donors (h‐MSCs), osteoblasts and endothelial cells. AML cells proliferation, immunophenotype and clonogenicity up to 21 days of 3D cultures have been analyzed. We characterized AML‐MSCs in comparison to h‐MSCs for proliferation rate, gene expression, secretome profile, osteogenic differentiation and anti‐inflammatory potential. We set up a drug targeting of the AML‐MSCs selecting agents among 480 compounds. We combined blasts and AML‐MSCs treatment in the 3D model. 3D‐AML cultures have been implanted in mice to generate robust pre‐clinical AML‐PDXs to study new combined treatments for AML. Results: We successfully set up 3D long‐term cultures of different primary AML (n = 20) and confirmed their proliferation up to 21 days. Clonogenic potential and immunophenotype of the original AML was also documented. We uncovered AML‐MSCs (n = 4) exhibiting an higher proliferation rate ( p &lt; 0.001) with respect to h‐MSCs (n = 2). AML‐MSCs (n = 8) were also found primed for osteogenic differentiation occurring after 7 days with respect to 21 days of h‐MSCs (n = 3, p &lt; 0.01). Gene expression profile of AML‐MSCs (n = 15) showed 513 genes up‐regulated with respect to h‐MSCs (n = 6) mostly related to pathways involved in cell proliferation and chromosome instability. HUVEC tube formation assay suggested that AML‐MSCs did not exert anti‐inflammatory activity in vitro , this latter supported by a peculiar secretome defined by mass‐spectrometry. AML‐MSCs drug screening identified 17 out of 480 active compounds for reducing AML‐MSCs proliferation without toxicity over h‐MSCs and AML blasts. Pilot studies of the 3D‐combined targeting of AML‐MSCs and AML blasts (with targeted compounds for genetic lesions) showed this strategy being dramatically synergic ( p &lt; 0.05) in vitro . In vivo , we obtained 4 different AML‐PDXs with different genetic high‐risk characteristics in a period ranging from 3 to 7 months for the first engraftment, and a median time of 2 months from P1 to P3. Mice were shown to maintain the original AML immunophenotype and genetic of diagnosis. Summary/Conclusion: We confirm long‐term cultures of AML as a suitable model for more predictable drug testing in vitro . Our humanized 3D niche was also demonstrated to potentiate the original leukemia engraftment in NSG mice. We are confident this findings supporting a reliable tool for more robust pre‐clinical studies that will enhance novel strategies for pediatric AML treatment.