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Persistence of myelofibrosis treated with ruxolitinib
found in ABL1, which involve a range of substitutions and directly alter binding of the drug with the enzyme.
What are the long-term outcomes for patients treated with ruxolitinib?
The COMFORT-1 study compared ruxolitinib with placebo for the treatment of myelofibrosis,38 and the COMFORT-2 study compared ruxolitinib with best avail- able therapy, including supportive care, hydroxyurea, and a range of other medical therapies.39 The primary endpoint in both studies was the proportion of patients achieving a 35% or greater reduction in spleen volume. The proportion of patients who achieved this response after 24 weeks of ruxolitinib treatment was 42% in COMFORT-1 and 32% in COMFORT-2.38,39 The response rates in the respective comparator arms of the two studies were 0.7% and 0%.
In a subsequent analysis of a subset of patients from the COMFORT-2 study the response rate after 48 weeks was 20% (confidence interval: 5.7-43.7%) in 20 patients with a CALR mutation versus 34% (confidence interval: 24.8- 44.1%) in 100 CALR-negative patients, most of whom had a JAK2 V617F mutation.37 Overall, there was no statistically significant difference in splenic response rate or survival between patients with and without JAK2 mutations, but the JAK2-positive patients had a numerically greater reduc- tion in spleen size.40 JAK2 V617F is almost universally pres- ent in post-polycythemia vera myelofibrosis, which is characterized by loss of heterozygosity and high allelic burden,41 and can perhaps be considered to epitomize JAK2-driven myelofibrosis. In this subgroup the hazard ratio for overall survival was 0.25, compared with 0.65 in primary myelofibrosis.40 Whether this reflects the higher frequency of high-risk genomic lesions in primary myelofi- brosis or ‘on-target’ efficacy through inhibition of JAK2 V617F remains to be established. Overall, these clinical data emphasize that ruxolitinib is not a JAK2 mutant-spe- cific therapy, although patients with CALR mutations might have mildly inferior responses.
Does ruxolitinib eradicate cells containing disease-causing driver mutations?
Although ruxolitinib is now established as the standard therapy for symptomatic myelofibrosis, evidence of a long-term effect on disease biology is limited. The average reduction in JAK2 allelic burden was 7-22% after 48 weeks of treatment in evaluable patients;38,42 regression of fibrosis in the marrow was seen in around 16% of patients at last follow-up (median 2.2 years),43 and there was no change in the risk of leukemic transformation. Cases of complete hematologic or molecular remission have been reported,44 but are very uncommon. Typically there is a gradual loss of response over time, with approximately 27% of patients remaining on first-line ruxolitinib treatment after 5 years in the COMFORT studies. In the subgroup of patients who achieved a 35% or greater reduction in spleen volume, the median duration of the response was 3.2 years.43,45
The immunophenotype of myelofibrosis-initiating cells has not been well studied, principally because of a lack of robust engraftment models, but in a few patients analyzed carefully, the stem cell population appeared to reside with- in the CD34+CD38–CD90+ compartment, as determined
using a humanized bone marrow niche.3 Flow cytometry studies suggest a high level of circulating CD34+CD38– hematopoietic stem-like cells in patients with mutant CALR, but all these cells were resistant to ruxolitinib in colony assays. New models of myelofibrosis will help to characterize the disease-propagating stem-like cells and identify critical cell surface markers for monitoring future disease-modifying therapies.
What are the possible mechanisms of ruxolitinib persistence?
A number of models have been proposed to explain rux- olitinib persistence, i.e., the ongoing outgrowth of mutated cells despite JAK-STAT pathway sensitivity to ruxolitinib, with varying levels of evidence. These include activation of alternative kinases not inhibited by ruxolitinib,46 epigenetic mutations leading to growth advantage,26,47 phosphatase negative feedback inhibition (as noted with the failure of RAFK inhibitors in melanoma),48 lack of specificity for JAK2 V617F versus wild-type JAK2,10 and accumulation of p-JAK2 during exposure to ruxolitinib. The last of these phenomena is the most studied, with multiple lines of evi- dence across several independent studies suggesting that it is a contributory mechanism in clinically observed ruxoli- tinib persistence.49-51 Various therapeutic strategies have been proposed in addition to targeting JAK2 (reviewed by Bankar and Gupta52). Here we focus on the evidence regarding persistent activation of JAK-STAT signaling and its downstream proteins, and how these phenomena may be targeted by current or emerging therapies.
Why does phosphorylation of JAK2 increase during ruxolitinib treatment?
Initial laboratory studies studying ruxolitinib activity in vitro noted that, paradoxically, phosphorylation of JAK2 on Tyr1007/1008 located in the activation loop was observed at increased levels in V617F-positive cells fol- lowing prolonged exposure to ruxolitinib.49 Both the total level of phosphorylation and the total amount of JAK2 protein were increased.49 Similar observations were also made for the type I inhibitor “JAK inhibitor 1” and Go6976, a protein kinase C inhibitor with potent activity against JAK2.53,54 Accumulation of p-JAK2 was noted to occur in the presence of total blockade of kinase function and inhibition of STAT and ERK phosphorylation down- stream (Figure 2). It was noted that ruxolitinib-induced phosphorylation of JAK2: (i) was staurosporine-sensitive and ATP-dependent; (ii) required cytokine receptor inter- action and intact JH1, FERM and JH2 domains; and (iii) could occur in V617F+ SET2 cells in the absence of JAK1 and TYK2.50 In contrast, JAK2 wild-type cells (such as TF- 1 cells) showed little or no type I inhibitor-induced loop phosphorylation after growth factor starvation. A car- boxyterminal-directed antibody could not immunopre- cipitate JAK2 after exposure to type I inhibitor. All these findings are consistent with a conformational change in the kinase domain generated by ruxolitinib. Because of presumed structural flexibility, high resolution crystallo- graphy data are not available for the activation loop dur- ing type I inhibitor binding but, critically, activation of downstream STAT phosphorylation could be repro-
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