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Targeting Myddosome Signaling In Published OnlineFirst August 20, 2018; DOI: 10.1158/1078-0432.CCR-17-3265 Cancer Therapy: Preclinical Clinical Cancer Research Targeting Myddosome Signaling in Waldenstrom's€ Macroglobulinemia with the Interleukin-1 Receptor-Associated Kinase 1/4 Inhibitor R191 Haiwen Ni1,2, Fazal Shirazi2, Veerabhadran Baladandayuthapani3, Heather Lin3, Isere Kuiatse2, Hua Wang2, Richard J. Jones2, Zuzana Berkova2, Yasumichi Hitoshi4, Stephen M. Ansell5, Steven P. Treon6, Sheeba K. Thomas2, Hans C. Lee2, Zhiqiang Wang2, R. Eric Davis2, and Robert Z. Orlowski2,7 Abstract Purpose: Waldenstrom's€ macroglobulinemia is an incur- apoptosis in cell lines and primary patient samples. Further able lymphoproliferative disorder driven by an L265P muta- downstream, R191 exposure led to reduced activation of tion in the myeloid differentiation primary response gene NF-kB, and of protein kinase B/Akt/mammalian target of 88 (MYD88), which activates downstream NF-kB signaling rapamycin signaling, whereas expression of a constitutively through the Myddosome. As this pathway depends in part on active Akt mutant induced R191 resistance. Gene expression activity of interleukin-1 receptor-associated kinases (IRAKs)-1 profiling and gene set enrichment analysis revealed a sig- and -4, we sought to evaluate the potential of the IRAK1/4 nature consistent with inhibition of c-Myc and activation of inhibitor R191 in preclinical models. the endoplasmic reticulum stress response. In both subcu- Experimental Design: Patient-derived cell lines and pri- taneous and systemic murine models of Waldenstrom's,€ mary samples were used in both in vitro and in vivo experi- R191 showed antitumor activity. Finally, the activity of ments to model Waldenstrom's€ macroglobulinemia and its R191 was enhanced when it was combined with novel response to IRAK1/4 inhibitors. chemotherapeutics such as bortezomib, afuresertib, and Results: R191 induced a dose- and time-dependent reduc- ibrutinib. tion in viability of BCWM.1 and MWCL-1 Waldenstrom's€ Conclusion: Taken together, these data support the trans- cell lines, and suppressed activation of IRAK1/4. This was lation of R191 as an approach to target IRAK1/4 to the € associated with cell-cycle arrest at G0–G1, reduced levels clinic for patients with Waldenstrom's macroglobulinemia. of cyclin-dependent kinases 4 and 6, and induction of Clin Cancer Res; 1–13. Ó2018 AACR. Introduction phoplasmacytic lymphoma and a serum monoclonal IgM para- protein (1, 2). Although some patients have asymptomatic Waldenstrom's€ macroglobulinemia is diagnosed in patients disease which can be followed without treatment, others present who have at least 10% bone marrow involvement with lym- with, or progress to symptomatic Waldenstrom's€ due to bulky disease, cytopenias, constitutional symptoms, or hyperviscosity, all of which require treatment. Chemotherapy for this disease 1Department of Hematology, The Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing, JangSu, China. 2Department of Lympho- has traditionally been based on the use of various combinations ma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, of drugs, including alkylating agents, anti-CD20 monoclonal Texas. 3Department of Biostatistics, The University of Texas MD Anderson antibodies, corticosteroids, nucleoside analogues, and protea- Cancer Center, Houston, Texas. 4Rigel, South San Francisco, California. 5Division some inhibitors (1, 2). These regimens typically are effective at 6 of Hematology, Mayo Clinic, Rochester, Minnesota. Dana Farber Cancer Insti- reducing disease burden and provide good long-term outcomes, 7 tute, Harvard Medical School, Boston, Massachusetts. Department of Experi- but complete responses are rare and Waldenstrom's€ is still mental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas. considered incurable. Understanding of the molecular pathobiology of Note: Supplementary data for this article are available at Clinical Cancer Waldenstrom's€ took a huge leap forward with the finding that Research Online (http://clincancerres.aacrjournals.org/). the vast majority of patients harbor an L265P mutation in the H. Ni and F. Shirazi contributed equally to this work. myeloid differentiation primary response gene 88 (MYD88; Corresponding Author: Robert Z. Orlowski, Department of Lymphoma and refs. 3, 4). MYD88 is recruited to activated Toll-like receptors Myeloma, The University of Texas MD Anderson Cancer Center, 1515 Holcombe (TLR) and interleukin-1 receptor (IL-1R; refs. 5, 6), resulting in Blvd., Unit 429, Houston, TX 77030. Phone: 713-794-3234; Fax: 713-563-5067; formation of MYD88 homodimers, which then interact with E-mail: [email protected] interleukin-1 receptor-associated kinase 4 (IRAK4) and IRAK2 doi: 10.1158/1078-0432.CCR-17-3265 to form the so-called Myddosome (7). Subsequent recruitment Ó2018 American Association for Cancer Research. to, and phosphorylation of IRAK1 by the Myddosome is www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 20, 2018; DOI: 10.1158/1078-0432.CCR-17-3265 Ni et al. effect on cannabinoid CB1-induced GTPgSbindinginCHO-K1 Translational Relevance cells, but did antagonize serotonin 5-HT2B-induced inositol Patients with Waldenstrom's€ macroglobulinemia are cur- monophosphate (IP1) release. R191 blocked IP1 release with rently treated with a variety of chemotherapeutics used alone an EC50 of 1.93 mmol/L, which may be due in part to cell or in combination, which have been adapted for use based on cytotoxicity, as R191 caused cytotoxicity in CHO-K1 cells with their activity in related disorders, including multiple myeloma an EC50 of 8.46 mmol/L, but R191 did not significantly inhibit a and indolent non-Hodgkin lymphomas. The first, and so far panel of related serotonin receptors. The effect of R191 was also only agent to receive regulatory approval in this disease is evaluated on the human potassium channel using human the Bruton's tyrosine kinase ibrutinib, but only a minority embryonic kidney (HEK) 293 cells transfected with a human of patients achieve a complete remission with this drug, Ether-a-go-go-related gene (hERG), and the maximal inhibition indicating a need for other rationally targeted therapies. This by R191 of the potassium-selective IKr current was 36.6% at 10 study provides evidence that the novel interleukin-1 receptor- mmol/L. With regard to selectivity, R191 was profiled against a associated kinase (IRAK)-1/4 inhibitor R191 has potent activ- panel of 76 kinases representative of the different subfamilies ity against preclinical and physiologically relevant models of of the full kinome. In addition to IRAK1 and IRAK4 (IC50 3 this disease. Because IRAK1/4 is a key signaling intermediate nmol/L), R191 inhibited the kinase activity of 11 kinases by downstream of the pathobiologic lesion responsible for >80% at 250 nmol/L and two kinases at 50 nmol/L. The Waldenstrom's,€ this supports the possibility that R191 could primary off-target activity of R191 was against Fms-related be an attractive option for translation to the clinic. tyrosine kinase (FLT)-3, which was confirmed in cellular assays [Flt3 ligand-induced extracellular signal-regulated kinase-1/2 phosphorylation and MV411 (Flt3-internal tandem duplica- tion) proliferation]. In addition, R191 inhibited the cellular required for binding of IRAK1 to tumor necrosis factor receptor– activity of additional targets (TAK1, AXL receptor tyrosine associated factor 6 (TRAF6), an E3 ubiquitin ligase that med- kinase, TANK binding kinase 1, and Bruton's tyrosine kinase) iates activation of the inhibitor of NF-kBkinase(IKK).Once at concentrations of 400 nmol/L or above, at least 20-fold IKK is activated, it phosphorylates the inhibitor of NF-kB(IkB), over its activity on IRAK1/4 signaling. prompting its ubiquitination and proteasome-mediated deg- Afuresertib, bortezomib, and ibrutinib were purchased from radation, freeing NF-kB to translocate to the nucleus and Selleck Chemicals. Other reagents were from Sigma-Aldrich activate its gene expression program (reviewed in ref. 7) to Corporation, unless otherwise indicated below. promote B-cell survival and proliferation (8–15). The L265P Cell lines and patient samples mutation within the MYD88 Toll/interleukin-1 receptor (TIR) € homology domain changes its structure and induces receptor The Waldenstrom's cell line BCWM.1 was a kind gift from Dr. Steven Treon (Dana Farber Cancer Institute, Boston, MA). activation-independent Myddosome formation, leading to À À þ þ þ À These cells are CD5 /CD10 /CD19 /CD20 /CD23 /CD27 / constitutive NF-kB activation (16). þ þ þ þ þ þ þ À € L265P CD38 /CD138 /CD40 /CD52 /CD70 /CD117 /cIgM /cIgG / In Waldenstrom's models, MYD88 forms complexes À À þ with and activates Bruton's tyrosine kinase (BTK), providing cIgA /ckappa ,andclambda , harbor the MYD88 L265P muta- a rationale for the use of tool compound BTK inhibitors, tion, and express low levels of wild-type CXCR4 (19). MWCL-1 Waldenstrom's€ cells were generously provided by Dr. Stephen which showed activity against preclinical models (3). These À þ Ansell (The Mayo Clinic, Rochester, MN), and are CD3 /CD19 / findings set the stage for testing of the BTK inhibitor ibrutinib, þ þ þ þ þ þ þ whose remarkable clinical activity against relapsed and/or CD20 /CD27 /CD38 /CD49D /CD138 /cIgM and ckappa . refractory Waldenstrom's€ (17) prompted its regulatory They also
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