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 To determine whether EPAG can offer a new therapeutic modality for hematopoietic regeneration in patients with IBMFS, we first sought to gain insights into the molecular mechanisms by which EPAG could enhance trilineage hematopoiesis in subjects with AA. Paradoxically, TPO levels are already markedly elevated in patients with severe AA; how adjunct therapy with the TPO mimetic EPAG could improve the stem cell deficit was thus a conundrum. Because marrow failure in AA largely results from the suppressive effects of IFN, we hypothesized that EPAG might support HSPC survival by activating signaling pathways in these cells distinctly from endogenous TPO under inflammatory conditions. To test this possibility, we directly compared 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 BM, thus providing an explanation for its clinical efficacy in AA, despite already elevated levels of TPO in these patients. This work was published Blood 133:2043-55 (2019). 1.2 Epag promotes DNA DSB repair in human HSPCs. To further elucidate the mechanisms by which EPAG improves hematopoiesis and assess its therapeutic potential in FA, we investigated whether EPAG could promote DNA repair in human HSPCs. We demonstrated that EPAG specifically activated the classical non-homologous end joining (C-NHEJ) DNA DSB repair mechanism, a pathway known to support genomic stability, in both normal human HSPCs and donor HSPCs subjected to CRISPR/Cas9-mediated knockdown of FANCA. Importantly, EPAG-mediated DNA repair resulted in enhanced genomic stability, survival, and function of primary human HSPCs, as demonstrated in karyotyping analyses, colony-forming unit (CFU) assays and after transplantation into immunodeficient NSG mice. This work was published Exp Hematol 73:1-6 (2019). Objective 2: Evaluate the safety and efficacy of Epag in subjects with Fanconi anemia. The ability of EPAG to stimulate multipotent long-term repopulating HSPCs in patients with AA, to maintain HSPCs under inflammatory conditions and to promote DNA repair in these cells suggested potential therapeutic relevance in FA-associated BMF. We have initiated an investigator-sponsored prospective phase I/II clinical trial of EPAG in Fanconi anemia (NCT03204188) and enrolled 19 patients to date (FANCA, n=15, FANCC, n=2, FANCE, n=1, and FANCG, n=1). One subject had MDS/monosomy 7 and was excluded at screening. Another patient withdrew early after identifying a suitable matched donor for transplant. All other subjects (n=17) received EPAG at an oral daily dose adjusted for age and ethnicity. 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 EPAG on an extension phase of the study for an additional 3 years. Fourteen (14) of the 17 evaluable patients have reached the 6-month assessment endpoint. A marrow response was observed in 13 subjects at 6 months; the marrow specimens were of poor quality at baseline and 6 months for one patient and response assessment could not be performed. Mean marrow cellularity increased by a factor of 3.0, and CD34+ HSPC frequency improved 1.8-fold at 6 months relative to pre-treatment values. Primary PB response was observed in 9 of 14 patients (64%) in one lineage (hemoglobin, n=4), two lineages (hemoglobin and platelets, n=3; hemoglobin and neutrophils, n=1) or three lineages (n=1) at 6 months of treatment. One patient who had a marrow but no PB response underwent an unrelated allogeneic HSPC transplantation and is clinically well. The other 13 subjects received EPAG on the extension phase of the study with stable or improving blood counts (range on study, 7 to 42 months). One patient who had stable but no increase in PB counts at 6 months displayed a unilineage response at 12 months and three subjects who displayed blood response in one or two lineages at 6 months met criteria for trilineage response at 9, 21 and 24 months on study. All evaluable patients tolerated the maximum dose of EPAG. A brief interruption was required for two subjects due to elevated liver function tests (LFTs). Liver abnormalities normalized within 7 to 14 days and treatment resumed without dose reduction. As frequently observed in Fanconi anemia, a similar LFT elevation was observed in both cases prior to EPAG treatment. Thus, attribution to the study drug cannot be confirmed. 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 (range, 1 to 6 months), affected patients initiated daily oral iron supplementation with gradual amelioration of iron depletion. To date, there has been no occurrence of marrow fibrosis, solid tumors or overt clinical transformation to MDS/AML. However, we observed monosomy 7 on standard metaphase analysis in one subject treated with EPAG for 12 months. There was no associated morphologic dysplasia or increased myeloblasts. Several studies have recognized cytogenetic abnormalities on BM examination of untreated FA patients, many of them noting a high frequency of monosomy 7. Thus, attribution of aberrant cytogenetics to EPAG cannot be validated in this patient. Nevertheless, given the well-documented association between monosomy 7 and increased risk for disease progression, EPAG was discontinued and he underwent an allogeneic stem cell transplantation. He is clinically well. Two patients died while on study. One subject had no improvement in neutrophil counts, developed Escherichia coli bacteremia and sepsis-related death occurred after 13 months of treatment with EPAG. The other patients demise was attributed to acute pulmonary hemorrhage in the context of severe refractory thrombocytopenia after 11 months on study. This work was presented at the American Society of Hematology (ASH 2019, ASH 2021) and the Fanconi Anemia Research Funds (FARF 2021, FARF 2022, FARF 2023).