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Persistence of myelofibrosis treated with ruxolitinib
mechanism of ruxolitinib persistence, its relationship to various recurrent somatic mutations in myelofibrosis, and potential ways to circumvent persistence to improve out- comes.
Physiological JAK-STAT signaling
JAK family kinases are non-receptor tyrosine kinases that are crucial for signal transduction of many cytokines and growth factors. The family comprises four members: JAK1, JAK2, JAK3, and TYK2.9 JAK family kinases are pre- associated with the cytoplasmic portion of their cognate receptors via their FERM and SH2 domains. Cytokine- induced receptor dimerization facilitates JAK kinase trans- activation and phosphorylation of tyrosine residues in the activation loop, as well as local phosphorylation of recep- tor cytoplasmic-tail tyrosine residues and tyrosine residues on associated signaling molecules. JAK2 contains a carboxy-terminal JAK homology domain, JH1, which has tyrosine kinase activity and transfers ATP to a protein substrate (such as STAT3 or STAT5), together with a JH2 pseudokinase domain, which is believed to regulate the activity of JH1.
Uncontrolled signaling by JAK2 is prevented by at least three major negative regulatory mechanisms. Activation loop phosphorylation of Tyr1007/1008 can be removed by tyrosine phosphatases including PTP1B, TC-PTP, SHP1, SHP2, CD45, and PTP-RT. Secondly, SOCS proteins (SOCS1-7 and CIS) are transcriptionally upregulated fol- lowing receptor activation and provide negative feedback loops that restrict the duration of active signaling by either directly inhibiting JAK or by promoting the degradation of the associated cytokine receptor.10-13 Thirdly, phosphory- lated JAK2 (p-JAK2) is ubiquitinated by CBL-family E3 ubiquitin ligases,14 as well as SOCS1, leading to proteaso- mal degradation, normally within minutes of receptor activation. Ubiquitination is itself a reversible process mediated by de-ubiquitinases, such as USP9X.15
Molecular aspects of JAK2 and ruxolitinib
The commonest somatic mutation in myelofibrosis is JAK2 V617F, which is present in 50-60% of patients with primary myelofibrosis and post-essential thrombo- cythemia myelofibrosis, and in 95% of patients with myelofibrosis following polycythemia vera.16 The V617F point mutation has been shown to disrupt the normal auto-inhibitory function of the JH2 domain leading to dys- regulated activation of JAK-STAT signaling which, both in animal models and in patients, contributes to many of the cardinal manifestations of the disease. Other mutations that occur in MPN may activate a cytokine receptor (e.g., thrombopoietin receptor) or downstream signaling pro- teins, including NRAS, KRAS, and PTPN11. The homod- imeric type 1 receptor for thrombopoietin regulates platelet formation and is encoded by MPL.
The exact molecular mechanism of JAK2 V617F, in com- parison with the action of wild-type JAK2 and other kinases such as JAK1 or TYK2, is still being elucidated. The molecular signature of this mutation is that it induces JAK autophosphorylation (activation) in the absence of cytokine. In vitro the presence of a homodimeric cytokine receptor is necessary for this to occur.17,18 Interestingly, the
JH2 domain of mutated JAK2 in myelofibrosis lacks an Asp in the His/Arg/Asp motif of its catalytic loop and pos- sesses no definitive kinase activity, even though it is required for cytokine receptor activation. The JH2 domain has, however, been shown to phosphorylate two negative regulatory residues of JAK2 (Ser523 and Tyr570), which may contribute to the increased kinase activity.19 A bio- chemical study showed that the orthologous mutation in JAK1 (JAK1 V658F, which has been found in patients with acute lymphoblastic leukemia and confers cytokine-inde- pendence in transduced Ba/F3 cells20,21) does not enhance the catalytic activity of the isolated kinase or indeed any other measurable enzymatic parameter.10 These findings are all consistent with the hypothesis that the V617F mutation does not render JAK2 more “active” when switched on but rather leads to its being switched on in inappropriate circumstances, such as in the absence of cytokine. Consistent with this, it was shown recently that JAK2 V617F promotes cytokine-independent receptor dimerization, thereby activating the kinase in the absence of an appropriate signal.22 Understanding the exact molec- ular effects of V617F will be important in the design of V617F-specific therapies that have lower activity against wild-type JAK2. Emerging in vitro and clinical data suggest that various JAK-STAT pathway-activating mutations (JAK2, MPL, SH2B3, NRAS, KRAS, PTPN11), as well as mutations in the protein scaffold CALR, all activate JAK- STAT signaling by subtly distinct mechanisms. This may have implications for treatment outcomes and suggests the existence of mutation-specific differences in ruxoli- tinib sensitivity and mechanisms of persistence.
More than 90% of cases of myelofibrosis show muta- tional evidence of JAK-STAT activation,16 suggesting that this pathway is a critical “necessary” driver of the patholo- gy. However, the converse is not true: having a mutation in the JAK-STAT pathway does not inevitably lead to myelofi- brosis, i.e., JAK2 V617F or mutant MPL is not sufficient for the disease phenotype. This is perhaps best exemplified by polycythemia vera, in which the JAK2 V617F variant allele frequency is commonly in the range 50-100%,23 yet pro- gression to myelofibrosis occurs in only 20-30% of individ- uals. This risk is time-dependent and was 15% with a median follow-up of 8 years in one study.24 Emerging stud- ies comprehensively detailing the genetic landscape high- light that myelofibrosis is a multi-mutation disease in the majority of patients.16 Importantly, mutations in epigenetic genes such as EZH2, ASXL1 or splicing factors as well the presence of specific inflammatory cytokines may link to fibrotic aspects of the pathology.25 In a large cohort of MPN patients, Grinfeld and colleagues performed an analysis to identify mutations that were associated with myelofibrosis versus chronic phase MPN. Five of the six genes with the highest odds ratio for myelofibrosis were epigenetic regula- tors (ZRSR2, U2AF1, SRSF2, EZH2, and ASXL1) and all had an odds ratio higher than that for JAK2 at an allele burden >50%.16 Although some of these mutations are likely acquired during disease evolution, there is also evidence that epigenetic changes early in MPN development may affect the phenotype. In a few patients with MPN who had mutations in both TET2 (which regulates DNA methyla- tion) and JAK2, the progenitor cells that appeared to have acquired TET2 mutations first were shown to be less sensi- tive to ruxolitinib than progenitors from other patients in whom JAK2 was acquired first.26 Determining the epigenet- ic mechanisms that contribute to ruxolitinib persistence/
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