Eltrombopag for the Treatment of Bone Marrow Failure Syndromes

National Heart, Lung, And Blood Institute

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Abstract

Objective 1: Characterize the mechanisms by which Epag promotes trilineage hematopoiesis. 1.1 Epag evades IFN blockade of signal transduction from c-MPL in human HSPCs Because TPO levels are already markedly elevated in subjects with SAA, we hypothesized that Epag might support HSPC survival by activating signaling pathways in these cells distinctly from endogenous TPO under inflammatory conditions. By directly comparing the effect of IFN on human HSPCs in the presence of TPO or Epag in vitro, we made two observations. First, we found that IFN perturbed TPO-induced signaling pathways in human HSPCs and that Epag could bypass this inhibition, resulting in enhanced progenitor activity and long-term HSPC repopulating potential in the presence of IFN. Second, we showed that IFN disrupted the low-affinity interaction between TPO and the extracellular domain of c-MPL by forming TPO-IFN heteromers, delineating a novel molecular mechanism by which IFN inhibits TPO signaling in HSPCs. Epag may bypass the inflammatory inhibition of signal transduction by avoiding capture by IFN in the bone marrow, thus providing an explanation for its clinical efficacy in SAA, despite already elevated levels of TPO in these patients. This work was published Blood 133:2043-55 (2019). Because erythropoietin (EPO), the primary regulator of red blood cell formation, shares significant sequence and structural homology with TPO, we also showed that IFN limits response of erythroid progenitors to EPO using a similar mechanism of EPO:IFN- heteromer formation, receptor binding occlusion and blunting of EPO signaling pathways. This understanding may facilitate the development of rational assays for high-throughput screening (HTS) of novel small molecules capable of overcoming the inflammatory block in anemia of inflammation (AI). A collaborative effort for HTS of small molecules for the treatment of AI is ongoing with investigators at the National Center for Advancing Translational Sciences (NCATS). This work was published Blood Cells, Disease, Molecules 85: 102488 (2020). 1.2 Epag promotes DNA DSB repair in human HSPCs. Recent investigations have uncovered a specific function of TPO in promoting non-homologous end joining (NHEJ)-mediated DNA double strand break (DSB) repair in HSPCs. To assess whether Epag may also promote DNA DSB repair in human HSPCs and to identify the pathways involved, CD34+ cells obtained from healthy individuals were cultured for 24 hours in the presence or absence of Epag and the DNA-PK inhibitor NU7441, exposed to low dose (2Gy) IR, and either transfected with NHEJ or HR DNA DSB repair reporter plasmids or assessed for changes in H2AX phosphorylation (H2AX), an indicator of IR-induced DSBs, at various times after IR. We demonstrated that Epag specifically activates the canonical NHEJ (C-NHEJ) DNA repair mechanism, a pathway known to support genome integrity. Importantly, Epag-mediated DNA repair resulted in enhanced genome stability, survival and function of primary HSPCs, as demonstrated in karyotyping analyses, CFU assays and after transplantation in immunodeficient NSG mice. Eltrombopag may thus offer a new therapeutic modality for the prevention of HSPC injury induced by IR in cancer therapy, and could have implications for the treatment of genome instability syndromes such as Fanconi anemia. This work was published Exp Hematol 73:1-6 (2019). Objective 2: Evaluate the safety and efficacy of Epag in subjects with Fanconi anemia. We have enrolled 10 patients (FANCA, n=9 and FANCC, n=1) to date in a prospective phase I/II study of Epag in Fanconi anemia (NCT03204188). The primary efficacy endpoint was the proportion of subjects with at least 2-fold increase in marrow cellularity or CD34+ HSPC frequency (marrow response), or clinically significant improvement in peripheral blood (PB) counts at 6 months (blood response). The primary safety endpoint was the global toxicity profile assessed at 6 months using CTCAE criteria. Responders were invited to continue on the extension phase of the study for an additional 3 years. Four of 10 patients have reached the 6-month assessment endpoint. A marrow response was observed in all 4 subjects at 6 months. Mean marrow cellularity increased by a factor of 4, and CD34+ HSPC frequency improved 2.5-fold at 6 months relative to pre-treatment values. Primary PB response was observed in 2 of 4 patients in one (hemoglobin) or two lineages (hemoglobin and platelets) at 6 months of treatment. Responders continue to receive Epag at 10 or 25 months with sustained improvements in blood counts and transfusion independence. The subject treated for 25 months recently met response criteria in a third lineage (neutrophils). Both patients who had a marrow but no PB response are clinically well; one subject underwent an unrelated allogeneic HSPC transplantation one year ago, and the other subject recently entered the extension phase of this study. No drug-related serious adverse events have occurred during Epag treatment. All patients tolerated the maximum dose of Epag. Patients without transfusional iron overload at study entry developed progressive depletion of iron stores attributable to the known potent iron chelating and mobilizing properties of Epag. At a median of 3 months after initiating Epag, affected patients initiated daily oral iron supplementation with gradual amelioration of iron store levels. To date, there has been no occurrence of marrow fibrosis, solid malignancies or clonal evolution. In sum, treatment with Epag was associated with increased marrow HSPCs in all subjects and PB responses in a subset of patients with FA. The overall safety profile was favorable. Further follow-up and expansion of the ongoing patient cohort will confirm Epags potential as a safe and efficient long-term therapeutic modality for FA-associated BMF. Alternatively, EPAG could also be used safely to boost marrow CD34+ cell numbers prior to autologous gene therapy applications. This work was presented at the American Society of Hematology (ASH 2019), the Fanconi Anemia Research Funds (FARF 2021) and submitted for presentation at the upcoming ASH Annual Meeting (ASH 2021). Objective 3: Evaluate the safety and efficacy of Epag in subjects with Diamond Blackfan anemia. In this study, we investigated whether Epag could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA induced pluripotent stem cell (iPSC) lines were generated from blood cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation with or without eltrombopag. DBA iPSCs exhibited early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic in DBA. This work was published Cells 10 (4): 734 (2021). In collaboration with the laboratory of Dr. Cynthia Dunbar (NIH), we have initiated a single-arm, pilot trial evaluating Epag in patients with steroid-refractory or steroid-intolerant DBA (NCT04269889). Fifteen subjects have been enrolled to date and response to treatment is under evaluation.