Differences in JAK Isoform Selectivity Among Different Types of JAK Inhibitors Evaluated for Rheumatic Diseases Through In Vitro Profiling.

OBJECTIVE: The selectivity of JAK inhibitors (Jakinibs) forms the basis for understanding their clinical characteristics; however, evaluation of selectivity is hampered by the lack of comprehensive head-to-head studies. Our objective was to profile in parallel Jakinibs indicated or evaluated for rheumatic diseases for their JAK and cytokine selectivity in vitro. METHODS: We analyzed 10 Jakinibs for JAK isoform selectivity by assaying their inhibition of JAK kinase activity, binding to kinase and pseudokinase domains, and inhibition of cytokine signaling using blood samples from healthy volunteers and using isolated peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis and from healthy donors. RESULTS: Pan-Jakinibs effectively suppressed kinase activity of 2 to 3 JAK family members, whereas isoform-targeted Jakinibs possessed varying degrees of selectivity for 1 or 2 JAK family members. In human leukocytes, Jakinibs predominantly inhibited the JAK1-dependent cytokines interleukin-2 (IL-2), IL-6, and interferons (IFNs). In PBMCs from patients with rheumatoid arthritis compared with healthy controls, inhibition of these cytokines was more pronounced, and some cell-type and STAT isoform differences were observed. Novel Jakinibs demonstrated high selectivity: the covalent Jakinib ritlecitinib showed 900- to 2,500-fold selectivity for JAK3 over other JAKs and specific suppression of IL-2-signaling, whereas the allosteric TYK2 inhibitor deucravacitinib inhibited IFNα signaling with high specificity. Interestingly, deucravacitinib targeted the regulatory pseudokinase domain and did not affect JAK in vitro kinase activity. CONCLUSION: Inhibition of JAK kinase activity did not directly translate into cellular inhibition of JAK/STAT signaling. Despite differences in JAK selectivity, the cytokine inhibition profiles of currently approved Jakinibs were highly similar, with preference for JAK1-mediated cytokines. Novel types of Jakinibs showed narrow cytokine inhibition profile specific for JAK3- or TYK2-mediated signaling.

Identification of Novel Small Molecule Ligands for JAK2 Pseudokinase Domain.

Hyperactive mutation V617F in the JAK2 regulatory pseudokinase domain (JH2) is prevalent in patients with myeloproliferative neoplasms. Here, we identified novel small molecules that target JH2 of JAK2 V617F and characterized binding via biochemical and structural approaches. Screening of 107,600 small molecules resulted in identification of 55 binders to the ATP-binding pocket of recombinant JAK2 JH2 V617F protein at a low hit rate of 0.05%, which indicates unique structural characteristics of the JAK2 JH2 ATP-binding pocket. Selected hits and structural analogs were further assessed for binding to JH2 and JH1 (kinase) domains of JAK family members (JAK1-3, TYK2) and for effects on MPN model cell viability. Crystal structures were determined with JAK2 JH2 wild-type and V617F. The JH2-selective binders were identified in diaminotriazole, diaminotriazine, and phenylpyrazolo-pyrimidone chemical entities, but they showed low-affinity, and no inhibition of MPN cells was detected, while compounds binding to both JAK2 JH1 and JH2 domains inhibited MPN cell viability. X-ray crystal structures of protein-ligand complexes indicated generally similar binding modes between the ligands and V617F or wild-type JAK2. Ligands of JAK2 JH2 V617F are applicable as probes in JAK-STAT research, and SAR optimization combined with structural insights may yield higher-affinity inhibitors with biological activity.

Male breast cancer: a survey at the Helsinki University Central Hospital during 1981-2006.

BACKGROUND: The purpose was to analyze the behavior of male breast cancer. METHODS: Fifty-eight male breast cancer patients were treated at the HUCH during 1981-2006. Data on risk factors, tumor characteristics, clinical presentation, treatment and survival were obtained by chart review. RESULTS: Presentation occurred at a median age of 63 years, most often due to a self-detected lump. The median size of the primary tumor was 1.8 cm and 14% were T4 tumors. Forty-seven percent had lymph node metastases and 4% distant metastases at diagnosis. Ductal carcinoma was the most common tumor type. All tumors with known receptor status were positive for estrogen receptor (ER) and 79% for progesterone receptor (PgR). Her-2 overexpression was found in 2/19 patients (11%). A family history of breast cancer, obesity, high alcohol intake and liver cirrhosis were the most often seen risk factors. Nineteen percent had one or two other malignancies, the most common second malignancy being prostate cancer in 7%. Ninety-seven percent were operated by mastectomy and 90% by axillary evacuation while sentinel node biopsy alone was done only in 7%. Sixty percent of the patients received radiotherapy, 64% adjuvant hormonal treatment, 20% adjuvant chemotherapy, and 2% adjuvant trastuzumab. Fourteen patients (25%) experienced a relapse of which 60% were distant, bone being the most common site. During follow-up 21 patients (37%) died, of whom nine of breast cancer and 12 due to other causes. The 5-year overall survival (OS) was 75%. CONCLUSIONS: Male breast cancer behaves and is today treated in many respects like postmenopausal breast cancer. However, due to rudimentary breast tissue the symptoms, diagnosis and especially a higher amount of T4 tumors differ from that of females. Also the risk factor profile and histologic subgroups seem different. The 5-year OS of 75% is clearly higher than 44% reported at our institution in 1982.