Placebo Controlled Functional Analysis of Eltrombopag in a Preclinical Xenograft Model of Myelodysplastic Syndromes (MDS)

Introduction: Thrombocytopenia is a common complication among MDS patients. Thus, many patients are dependent on platelet (PLT) transfusions, which give short-term therapeutic relief but are also associated with considerable clinical risks. In this context, thrombopoietin receptor agonists (TRAs) are under investigation as alternative treatment option, albeit with the concern that these substances may promote adverse events in MDS. However, beside potential positive effects on thrombopoiesis in MDS patients the TRA Eltrombopag (EPAG) has also been shown to exert positive disease modifying effects in vitro (Roth et al., Blood 2012). Using a MDS xenograft model, we here investigate the efficacy of EPAG and its influence on clonal composition on primary patient derived MDS xenografts and present data from an ongoing study.

Methods: Currently, samples from n=18 MDS patients (MDS del(5q)=2, MDS-MLD=6, MDS-RS-MLD=1 MDS-EB-1=2, MDS-EB-2=7) have been xenografted into NSG mice by intrafemoral co-injection of CD34⁺ hematopoietic stem cells and mesenchymal stromal cells using a modified protocol according to Medyouf et al., Cell Stem Cell 2014. Long term engraftment is assessed 12 weeks post-transplant by intrafemoral bone marrow (BM) biopsy and mice with positive human engraftment are subsequently treated with either EPAG (50mg/kg) or vehicle control for 18 weeks. During that time, the mice are bled every two weeks and BM aspiration is performed every six weeks. Human hematopoietic cells are FACS sorted. In peripheral blood, human PLTs are specifically and absolutely counted with a FACS assay based on hCD41⁺ cells and beads. To track clonal composition of MDS samples upon xenografting and EPAG treatment in comparison to placebo control, the original patient sample and the final MDS xenograft sample are being whole exome sequenced (WES). Interspersed time points are analyzed with a patient individual amplicon based deep sequencing approach (Mossner et al., Blood 2016) to calculate dynamics of variant allele frequencies (VAF) in dependency of treatment.

Results: To date, n=12 patient samples have been analyzed for human engraftment after 12 weeks post-transplant. Of these, n=7 (58%) have shown positive human engraftment and are being treated with EPAG versus placebo. To this end, one case has been completely followed up, including final molecular analysis. This MDS high risk case (MDS-EB-2) with a clinical PLT count of 29x10⁹ PLT/L was transplanted into n=3 NSG mice. While two mice treated with EPAG survived the complete duration of the experiment, the placebo mouse died prematurely due to severe weight loss after 6 weeks of treatment. Further, EPAG treatment led to an initial rise of human PLT levels, while the placebo treated mouse presented a continuous decline of human PLTs, showing the efficacy of EPAG on human xenografts in the model. This observation has been confirmed in another case currently still under treatment. Molecular tracking by WES confirmed MDS patient specific molecular lesions in the MDS xenograft such as monosomy 7 and the disease related mutations CBL, DNMT3A and EZH2 with VAFs of 83%/43%/23% respectively. The monosomy 7 was detectable in all mice. CBL and DNMT3A exhibited similar VAFs in mouse EPAG1 (VAF=100%/54%), EPAG2 (VAF=100%/34%) and placebo (VAF=100%/50%). The EZH2 mutation was only detected in mouse EPAG2 (VAF=11%). Interestingly, the placebo mouse acquired a de novo mutation of U2AF1 (VAF=10%), which was not detectable in the initial patient sample or the EPAG treated mice. This spliceosomal mutation is associated with a higher risk of transformation to AML and shorter survival (Graubert et al., Nat Genet 2012; Makishima et al., Blood 2012).

Conclusions: Our data show first proof of principle results that new treatment options can be tested successfully in a preclinical murine xenograft model of primary MDS patient samples in a placebo controlled experimental setting. This approach allows the performance of patient individual substance testing that can segregate substance specific effects from natural disease progression in the same patient. Clinical parameters such as human PLT production and molecular clonal composition can be measured with a high confidence in vivo. Our current data show preliminary support for the hypothesis that EPAG may be efficacious in increasing PLT production in MDS patients without adversely influencing the underlying clonal composition.