Interim translational data from the MANIFEST phase II study of pelabresib in myelofibrosis

Myelofibrosis (MF) is characterized by abnormal megakaryopoiesis and overproduction of proinflammatory cytokines, which lead to bone marrow fibrosis, progressive anemia, and extramedullary hematopoiesis resulting in hepatosplenomegaly. Bromodomain and extraterminal domain (BET) proteins are involved in regulating neoplastic myeloproliferation and proinflammatory cytokine expression mediated by nuclear factor kappa B (NF-κB). Therefore, BET proteins are an attractive target for drug development in MF.

Pelabresib is a potent and selective BET bromodomain inhibitor currently under investigation for the treatment of MF. The MPN Hub has previously reported on the phase II MANIFEST clinical trial (NCT02158858) of pelabresib in MF. At the 26th Congress of the European Hematology Association (EHA2021), Patricia Keller and colleagues, presented the interim translational data from the MANIFEST trial.¹ The results are summarized below.

Study design

Phase II, open-label clinical trial of pelabresib as monotherapy, or in combination with ruxolitinib. The study had three arms, broken down into five cohorts depending on transfusion dependence (Table 1), with different clinical endpoints for each cohort.

Table 1. MANIFEST trial design*

JAKi, JAK inhibitor; TD, transfusion dependent; TI, transfusion independent. *Adapted from Keller et al.1
Arm	Eligibility	Treatment	Cohort
1	Prior treatment with ruxolitinib, but patients are refractory/intolerant/ineligible	Pelabresib	1A: TD 1B: non-TD
2	Current treatment with ruxolitinib but with suboptimal response or MF progression	Pelabresib with ruxolitinib	2A: TD 2B: non-TD
3	JAKi naïve	Pelabresib with ruxolitinib	JAKi-naïve

The clinical endpoints for each cohort were as follows:

• Cohort 1A: Transfusion dependent to transfusion independent
• Cohort 1B: Spleen volume reduction of 35% (SVR35)
• Cohort 2A: Transfusion dependent to transfusion independent
• Cohort 2B: SVR35
• Cohort JAK inhibitor (JAKi)-naïve: SVR35

The pharmacodynamic (PD) activity of pelabresib was assessed including changes in inflammatory cytokines in the blood, improvement of bone marrow biology including bone marrow fibrosis (BMF), and impact of mutation status on clinical response:

• Measurement of interleukin-8 (IL-8; CXCL8) gene expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) at pre-dose and post-dose on Day 1 of Cycle 1.
• Analysis of plasma cytokine levels at baseline compared with healthy donors, and response of cytokines to treatment at 14 days, and longitudinally at 6, 12, and 24 weeks post-treatment.
• BMF grade by reticulin fibrosis staining on bone marrow biopsy was performed at baseline and 24+ weeks post-treatment.
• Exploratory immunohistochemistry (IHC) analysis of erythroid progenitors and megakaryocyte numbers, and clustering by staining for CD71 (erythroid) and CD61 (megakaryocyte), was performed at baseline and 24 weeks.
• Ex vivo differentiation of isolated CD34+ cells from baseline patient samples with erythroid differentiation-promoting cytokines.
• Next-generation sequencing (NGS) was conducted using DNA from peripheral blood for mutation profiling.

Results

Rapid reduction of IL-8 (CXCL8) gene expression as a PD marker

• A rapid PD response was demonstrated by a 55% median reduction of the blood IL-8 mRNA level compared with baseline, 4 hours after the first dose of pelabresib in 102 patients, with similar reduction across arms.
• Only 16/102 patients had less than 10% decrease or increase in IL-8 expression 4 hours post-treatment.

Pelabresib plus ruxolitinib reduced levels of cytokines linked to inflammation and NF-κB

• At baseline, NF-κB and inflammation-related cytokine levels were elevated across all patients in the MANIFEST trial when compared with six healthy controls.
• Pelabresib and its combination with ruxolitinib reduced the levels of cytokines linked to inflammation and NF-κB from 14 days post-treatment, and through Week 24 of treatment.
• Several JAKi-regulated cytokines were more strongly downregulated in the JAKi‑naïve Arm 3 patients than the ruxolitinib-intolerant or -refractory patients in Arms 1 and 2.
• Several cytokines not previously reported to be regulated by JAKi were downregulated in both pelabresib monotherapy and combination therapy groups.

Improvements in BMF and erythroid and megakaryocyte populations

• A BMF central review was performed in 63 patients at the time of analysis.
• In total, 83% of patients demonstrated ≥1 grade improvement in BMF at 24 weeks, with four patients achieving at least Grade 2 improvement.
• IHC analysis by a central pathologist review was performed for 37 patients at the time of analysis.
• An increase in BM erythroid progenitors was observed in 59% of patients, and a decrease in BM megakaryocyte numbers and/or clustering was observed in 65% of patients (Table 2).

Table 2. Improvement in BM erythroid progenitors and megakaryocyte numbers and clustering*

Eryth, erythroid progenitors; MK, megakaryocyte. *Adapted from Keller et al.1
Cell population	Arm 1 n = 8	Arm 2 n = 17	Arm 3 n = 12	Total N = 37
Eryth (CD71+), %	38	65	67	59
MK (CD61+), %	38	59	92	65

Pelabresib promotes early erythroid differentiation, and partially reverses ruxolitinib-mediated suppression of early-stage erythroid differentiation ex vivo

• Pelabresib was shown to promote early erythroid differentiation from colony-forming unit-erythroid cells (CD34+ CD71+) to proerythroblast cells (CD34− CD71+) to a similar degree in both healthy donors and MF patients.
• Pelabresib partially reverses the effects of ruxolitinib on early erythroid differentiation.

Mutation analysis showed similar mutation profiles across study arms

• Similar mutational patterns were seen across the three arms of the study, with the most frequently mutated genes in:
  • JAK2: 65% of patients
  • ASXL1: 47% of patients
  • TET2: 23% of patients
  • CALR: 18% of patients
• No significant differences in SVR35 response at 24 weeks were observed in patients with or without single gene mutations in the top 14 mutated genes at baseline (all arms, n = 150 total patients assessed).
• Patients with ASXL1 mutations (associated with a poor prognosis and higher risk for AML transformation) showed similar SVR35 response rates at 24 weeks to those without.

Conclusion

The interim translational data from the MANIFEST study demonstrate a robust PD effect of pelabresib in patients with MF. Pelabresib monotherapy, and in combination with ruxolitinib, impacted both cytokine expression and bone marrow function and showed broad clinical activity regardless of mutational status. MANIFEST-2 (NCT04603495) is a global, randomized, double-blind phase III trial that is currently underway to evaluate the combination of pelabresib with ruxolitinib versus placebo with ruxolitinib in JAKi-naïve patients with MF.