ALBERT

Description: One Japanese pedigree of familial essential thrombocythemia (FET) inherited in an autosomal-dominant manner is presented. A unique point mutation, serine 505 to asparagine 505 (Ser505Asn), was identified in the transmembrane domain of the c-MPL gene in all of the 8 members with thrombocythemia, but in none of the other 8 unaffected members in this FET family. The Ba/F3 cells expressing the mutant Asn505 acquired interleukin 3 (IL-3)-independent survival capacity, whereas those expressing wild-type Ser505 did not. The autonomous phosphorylation of Mek1/2 and Stat5b was observed in the mutant Ba/F3 cells in the absence of IL-3. The former was also found in platelets derived from the affected individual in the absence of thrombopoietin. These results show that the Asn505 is an activating mutation with respect to the intracellular signaling and survival of the cells. This is the first report of FET deriving from a dominant-positive activating mutation of the c-MPL gene. (Blood. 2004;103: 4198-4200)

Description: Key Points A mutation preventing interaction between c-Myb and p300 prevents transformation and leukemia induction by MLL-AF9 and AML1-ETO9a oncogenes. Identifying agents that block the c-Myb-p300 interaction may be a valuable approach to developing a therapy for acute myeloid leukemia.

Description: Introduction: Thymus and activation-regulated chemokine (TARC/CCL17) and macrophage derived chemokine (MDC/CCL22) are specific ligands of CC chemokine receptor 4 (CCR4). We have previously reported that tumor cells obtained from a large majority of patients with adult T-cell leukemia/lymphoma (ATLL) express CCR4 and that the extent of CCR4 expression is significantly associated with unfavorable prognosis and with skin involvement. We also reveal TARC/CCL17 and MDC/CCL22 secreted by tumor cells create a favorable environment for tumor escape from host immune defense withattract CCR4+ regulatory T cells to the tumor. Collectively, it is assumed that the interactions between CCR4 and its ligands play an important role for pathogenesis of ATLL. The goal of the present study was to clarify the clinical roles of individual CCR4 ligands in ATLL patients. Material and Method: We compared the serum levels of CCR4 ligands (TARC/CCL17 and MDC/CCL22) and helper T-cell associated cytokines (IFN-g, TNF-a, IL-2, IL-4, IL- 5, IL-6, IL-10), in 103 ATLL patients, 40 asymptomatic HTLV-1 carriers, and 50 healthy adult volunteers. We also analyzed the associations between the serum concentration of individual CCR4 ligands and the clinical characteristics such as survival time among the ATLL patients. Result: Serum levels of TARC/CCL17 and MDC/CCL22 were significantly lower both in ATLL patients and asymptomatic HTLV-1 carriers than in healthy controls. The overall survivals were significantly shorter in ATLL patients with a high TARC/CCL17 level than in those with a low TARC/CCL17 level. Univariate cox proportional hazard analysis identified the 12 unfavorable prognostic factors including a high TARC/CCL17 level as well as a high IL-5 level, a high IL-6 level, and a high IL-10 level(Table). Multivariate analysis confirmed that a high TARC/CCL17 level was an independent and significant unfavorable prognostic factor among the ATLL patients (Table). Although a high MDC/CCL22 level was significantly associated with a high TARC/CCL17 level, serum MDC/CCL22 levels did not have prognostic significance in ATLL patients. Conclusion: Measurement of serum TARC/CCL17 levels is useful for predicting the prognosis and for determining a suitable treatment strategy for ATLL patients. Prognostic factors affecting overall survival in all ATLL patients. (n=103) Variable Unfavorable Unlvarlate Multivariate Hazard ratio (95%*CI P value Hazard ratio (95%*CI) P value *CI, confidence Interval TARC/CCL17 〉 285 pg/mL 1.638 (1.022–2 626) .0405 1.814 (1.109–2.969) .0177 IL-5 〉 1.7 pg/mL 2.307 (1.357–3.922) .0020 1.767 (1.008–3.096) .0468 IL-6 〉 0.7 pg/mL 1.956 (1.183–3.233) .0089 - IL-10 〉 2.6 pg/mL 2.192 (1.254–3.830) .0059 - Clinical subtype Acute, lymphoma 3.539(1.851–6.767) .0001 - PS 2–4 2 187 (1.372–3.488) .0010 - B symptoms Present 2.104 (1.279–3.461) .0034 1,729 (1.040–2.872) .0346 Hemoglobin 〈 100 g/dL 1.923 (1.030–3.592) .0401 - Albumin 〈 3.0 g/dL 1.854(1.029–3.338) .0397 - LDH 〉 2N 2.683(1.683–4.279) 〈 .0001 1.876(1.086–3.242) .0241 sIL-2R 〉 10,000 U/mL 3.232(1.896–5.507) 〈 .0001 1.956(1.024–3.738) .0423 Extranodal involvement ≥ 2 2.152 (1.352–3.426) .0012 -

Description: Purpose: ADCC is a major antitumor mechanism for the action of therapeutic monoclonal antibodies (mAbs) such as rituximab, trastuzumab and cetuximab. Therefore, a better understanding of ADCC will allow the development of novel, more effective treatment strategies, and may help overcome the resistance which can develop against the effects of the therapeutic mAbs. However, the tumor-associated factors which determine susceptibility to rituximab-induced ADCC have not been identified. In the present study, we focused on this issue, especially focused on the molecules expressed by the tumor cells that interact with NK cells, such as NKG2D ligands, because of the importance of NK cells for rituximab-induced ADCC. The aim of this study was to identify tumor associated factors which determine susceptibility to rituximab-induced ADCC. Experimental Design: 30 different CD20+ non-Hodgkin lymphoma (B-NHL) cell lines were phenotyped for characteristics of cell surface protein: expression levels of CD20, MHC class I, NKG2D ligands (ULBP1-3, MICA, and MICB), CD48 (2B4 ligands), HLA-G, cathepsin B, and complement inhibitors (CD46, CD55, and CD59), and the influence thereof on susceptibility to rituximab-induced ADCC was established. Result: The degree of rituximab-induced ADCC were correlate with the expression levels of CD20 and ULBP1-3, and inversely correlate with the expression levels of MHC class I among 30 different CD20+ B-NHL cell lines. The importance of the ULBPs was confirmed using antibody blockade. In the presence of blocking mAb to ULBP1, 2 or 3, a decrease of rituximab-induced ADCCs against B-NHL cell lines were observed. In addition, the present study clearly identified the key mechanism of rituximab-induced ADCC as antibody-dependent target-specific cytotoxicity mediated by highly activated NK cells. Strong NK cells activation was due to the combination of Fc„dR stimulation via the Fc portion of rituximab, together with stimulation of activating NK cell receptors via their ligands expressed on the tumor cells, particularly ULBPs, which occurred in a robustly synergistic manner. Conclusions: Tumor cell susceptibility to rituximab-induced ADCC was determined by three major tumor-associated factors: the amount of the target molecule, CD20; the amount of the ligands for inhibitory killer Ig-like receptors, MHC class I; and the amounts of some of the NKG2D ligands, especially ULBP1-3. This is the first report to show the importance of ULBPs on tumor cells for rituximab-induced ADCC. The ULBPs could be valuable diagnostic biological makers and significant targets for immunotherapy to improve efficacy not only of rituximab but also of other therapeutic mAbs. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel.

Description: The molecular pathogenesis in chronic myeloproliferative disorder including essential thrombocythemia (ET) has been recently clarified. We previously identified a novel activating mutation of c-MPL gene (S505N) as a cause of familial ET. The Jak2 mutation (Jak2V617F) is also reported most frequently as a constitutive activating mutation in ET. Here we screened the mutations in the open reading frames of thrombopoietin (TPO), c-MPL and Jak2 in the 26 Japanese (sporadic) ET patients. None had any somatic mutations in TPO. We found Jak2V617F mutation in 7 patients. Interestingly, one of the patients had the c-MPL somatic mutation (W515L). The c-MPL W515L transfectants presented the autonomous STAT5 and MEK1/2 phosphorylation in the absence of the ligand, which show the c-MPL W515L is a constitutive activating mutation for signalling. The concurrent mutations of Jak2V617F and c-MPL W515L are likely to contribute to the pathogenesis of the thrombocythemia in this patient. Now we are focusing on the issue whether the single clone with both of two mutations occupy or the two clones with either Jak2 or c-MPL mutation coexist in the bone marrow of this ET patient (Single cell PCR is on going with the megakaryocytes of this patient).

Description: The c-MPL gene and its ligand, thrombopoietin (TPO), regulate the proliferation and differentiation of megakaryocytes and platelets. We previously found a dominant-positive activating mutation of the c-MPL gene as a cause of familial essential thrombocythemia. This mutation (Ser505 to Asn505) was located in the transmembrane domain of the c-MPL. The Ba/F3 cells expressing the mutant allele of Asn505 acquired interleukin-3 (IL-3)-independent survival capacity and the autonomous phosphorylation of Mek1/2 and Stat5b in the absence of IL-3 (Blood 103; 4198, 2004). The mechanism underlying how c-Mpl with Asn505 induces constitutive phosphorylation of Mek and Stat5b remains unknown. However, it is assumed that dimerization of c-Mpl molecules may be accelerated as a result of this specific mutation, as in the cases of mutations of c-kit and Neu at their transmembrane domains. Thus we examined the status of dimer formation of the c-Mpl molecules in the Ba/F3 cells with the Asn505 cells in the presence and absence of IL-3 or Tpo using the cell surface chemical cross-linking assay. As a result, in wild-type (Ser505) c-Mpl-expressing Ba/F3 cells, homodimeric signals of the c-Mpl proteins were detected only in the presence of IL-3 or Tpo, whereas mutant (Asn505) c-Mpl-expressing Ba/F3 cells harbored constitutively homodimeric signals even in the absence of IL-3 or Tpo. These observations suggest that mutant c-Mpl with Asn505 can activate the downstream signals involved in the Tpo-c-Mpl signaling pathway mediated by the formation of autonomous homodimerizaton independent of ligand stimulation. As asparagine (Asn) is well known to provide a strong free energy of association by forming interhelical hydrogen bonds in a hydrophobic environment, the Asn505 mutation in the transmembrane domain of the c-MPL is thought to drive strong interactions (dimerizations) among the c-Mpl molecules, leading to the activation of the Tpo-c-Mpl signaling and consequently autonomously activated megakaryocytopoiesis with the excessive production of platelets. The disulfide-bonded homodimerization may be also induced by the Asn505 mutation. Further investigation of this issue is now in progress.

Description: We previously reported that a dominant-positive activating mutation (Asn505) in the transmembrane domain (TMD) of c-MPL, which encodes the thrombopoietin receptor, caused familial essential thrombocythemia. Here, we show that the Asn505 mutation induces both autonomous dimerization of c-Mpl and signal activation in the absence of its ligand. Signal activation was preserved in a truncated mutant of Asn505 that lacked the extracellular domain of c-MPL. We also found that the substitution of the amino acid (AA) residue at position 505 with others of strong polarity (Glu, Asp, or Gln) also resulted in activated dimerization without ligand stimulation. Overall, these data show that the Asn505 mutation transduced the signal through the autonomous dimerization of the c-MPL protein due to strong AA polarity. This finding provides a new insight into the mechanism of disease causation by mutations in the TMD of cytokine/hematopoietic receptors.

Description: Bortezomib, a proteasome inhibitor, which was originally developed as an inhibitor of NF-kappaB pathways, is currently used for the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL). The mechanisms of action of this anti-tumor agent have been studied by several investigators. As bortezomib has been reported effective for a fraction of peripheral T-cell lymphomas including adult T-cell leukemia/lymphoma (ATLL) and cutaneous T-cell lymphoma (CTCL) at clinically achievable concentrations, it is expected to be applicable for these refractory T-cell malignancies. In this study, we have explored the underlying mechanisms of bortezomib-induced apoptosis in cell lines established from CTCL and ATLL together with those from MM by particularly focusing on the level of mitochondrial membrane injury. It was because the apoptosis induced by bortezomib in CTCL and ATLL cells accompanied the activation of caspase-3, -8, and -9, and the disturbance of mitochondrial membrane potential preceded the process of bortezomib-induced apoptosis when detected by DiIC1 and Annexin V staining. In 6 MM lines including KMS-12-PE, 2 CTCL lines including Hut78, and 7 ATLL lines including ATN1 and MT4, anti-apoptotic factors such as c-Flip and XIAP were down-regulated after exposure to bortezomib, probably via inhibition of NF-kappaB signaling. This reduction of c-Flip and XIAP was also confirmed in the primary tumor cells derived from the patients with ATLL after exposure to 20nM bortezomib. These findings indicate that the activation of both intrinsic and extrinsic apoptosis pathways led to activation of caspases via reduced expression of negative regulators of apoptosis. Among the members of the BH3-only family protein, up-regulation of Noxa was consistently seen at both the transcriptional and protein levels in a p53-independent and c-Myc-independent manner after exposure to bortezomib, whereas the expression of other BH3-only family proteins showed no consistent changes. Of anti-apoptotic Bcl-2 family proteins, accumulation of Mcl-1 and its cleaved form were observed, but no altered expression of other Bcl-2 family members was seen. Specific repression of Noxa by small interfering RNA partially rescued CTCL and ATLL cells from bortezomib–induced apoptosis, suggesting a crucial role of Noxa in bortezomib-induced apoptosis in these cell types as well as in MM cells. Immunoprecipitation assays indicated time-dependent binding of Noxa and Mcl-1 in all cell types, suggesting that functional repression of Mcl-1 led to the loss of mitochondrial outer membrane potential. Similar results showing transcriptional and translational up-regulation of Noxa followed by increased amount of the Mcl-1/Noxa complexes after exposure to 20nM bortezomib were also obtained in primary tumor cells derived from patients with ATLL. Taken together, we conclude that bortezomib-induced apoptosis in ATLL/CTCL cells at least partly depends on up-regulation of Noxa and functional repression of Mcl-1 in addition to the reduced amount of c-Flip and XIAP proteins, although the mechanisms leading to the transcriptional activation of Noxa after exposure to bortezomib remains to be clarified.