Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia

Abstract

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) comprises over half of Philadelphia chromosome-like (Ph-like) ALL, is associated with poor outcome in children and adults. Overexpression of CRLF2 results in activation of JAK-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of Janus kinases show variable and limited efficacy. Here we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against Janus kinases. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of multiple series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded Janus kinases and potently killed CRLF2­-rearranged cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1, and suppressed proliferation of CRLF2-rearranged ALL in vivo. While dual JAK/GSPT1-degrading PROTACs were most potent, development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading GSPT1-sparing PROTAC that demonstrated efficacy in the majority of the kinase-driven xenografts which were otherwise unresponsive to type I JAK inhibitors. Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL, and highlight the interplay of Janus kinase and GSPT1 degradation activity in this context.

JAK inhibitors and COVID-19

“… 92 Only in 2021, in a very recent study which is being published in Blood the structures of ruxolitinib and of baricitinib were reported with the JAK2 kinase domain. 93 Of great interest, these structures allowed the first examination of ruxolitinib and baricitinib engaged with their main target ( figure 2B ). It also afforded design of ruxolitinib and baricitinib modified molecules where a linker was introduced to a solvent exposed carbon (C2 of the pyrimidine ring) of each inhibitor to which pomalidomide or thalidomide were linked.…”

Biologic and Therapeutic Implications of Genomic Alterations in Acute Lymphoblastic Leukemia

“…Combinatorial use of kinase inhibitors against multiple signaling has shown synergism in patient-derived xenograft (PDX) models of CRLF2/JAK mutant (JAK and PI3K/mTOR inhibitors), ABL/PDGFR mutant (dasatinib and PI3K/mTOR inhibitor) and EPOR-rearranged (ponatinib and ruxolitinib) [119]. Moreover, recently dual JAK/GSPT1-degrading proteolysis-targeting chimeras PROTACs have been developed and showed efficacy in Ph-like B-ALL kinase-driven PDX models which were otherwise unresponsive to type I JAK inhibitors [120]. Lastly, the use of immunotherapeutic agents, such as blinatumomab, inotuzumab, and CAR-T cells (including those targeting CRLF2 [121], represents a promising alternative approach for this subtype which is irrespective of a specific genetic alteration or response to prior chemotherapies [104]).…”

Preclinical studies of Flonoltinib Maleate, a novel JAK2/FLT3 inhibitor, in treatment of JAK2V617F-induced myeloproliferative neoplasms

“…As marketed drugs for the treatment of MPNs, ruxolitinib is a JAK1/JAK2 inhibitor, and fedratinib is a JAK2/FLT3 inhibitor. Both of them bind to the JAK2 JH1 domain, which may be the reason why their selectivity is not sufficiently high, and they cause significant side effects [ 32 , 33 ]. JAK2 V617F mutation is located in the JAK2 JH2 domain.…”

“…The V617F mutation site is located above the N-terminus of the JH2 domain [ 27 ], and when the valine at position 617 is replaced by phenylalanine with a higher molecular weight, the prolongation of helix C and the interaction between F617 and the helix C phenylalanines 594 and 595 of JH2 domain is stable and activates the JH1 domain [ 28 – 31 ]. Ruxolitinib and fedratinib are the most representative JAK kinase inhibitors for the treatment of MF, and both of them bind to the JAK2 JH1 domain [ 32 , 33 ]. Recently, Bristol Myers Squibb Company (BMS) has developed a highly selective TYK2 inhibitor, named BMS-986165, which acts by binding to the TYK2 JH2 domain.…”