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The caspase recruitment domain family member (CARD)11-Bcl10-Malt1 signalosome controls TGF-β–activated kinase 1 (TAK1) activation and regulates BCR-induced NF-κB activation. In this study, we discovered that CARD19 interacted with TAK1 and inhibited TAB2-mediated TAK1 ubiquitination and activation. Although CARD19 deficiency in mice did not affect B cell development, it enhanced clonal deletion, receptor editing, and anergy of self-reactive B cells, and it reduced autoantibody production. Mechanistically, CARD19 deficiency increased BCR/TAK1-mediated NF-κB activation, leading to increased expression of transcription factors Egr2/3, as well as the E3 ubiquitin ligases c-Cbl/Cbl-b, which are known inducers of B cell tolerance in self-reactive B cells. RNA sequencing analysis revealed that although CARD19 deficiency did not affect the overall Ag-induced gene expression in naive B cells, it suppressed BCR signaling and increased hyporesponsiveness of self-reactive B cells. As a result, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus. In summary, CARD19 negatively regulates BCR/TAK1-induced NF-κB activation and its deficiency increases Egr2/3 and c-Cbl/Cbl-b expression in self-reactive B cells, thereby enhancing B cell tolerance.

PDF file - 251K, Chemical structures of ganetespib and crizotinib.

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PDF file - 486K, The addition of ganetespib to crizotinib does not result in added toxicity in H3122 xenografts.

PDF file - 2538K, Crizotinib-resistant H3122 CR1 cells express an EMT phenotype.

PURPOSE Lehmann et al have identified four molecular subtypes of triple-negative breast cancer (TNBC)—basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor—and an immunomodulatory (IM) gene expression signature modifier. Our group previously showed that the response of TNBC to neoadjuvant systemic chemotherapy (NST) differs by molecular subtype, but whether NST affects the subtype was unknown. Here, we tested the hypothesis that in patients without pathologic complete response, TNBC subtypes can change after NST. Moreover, in cases with the changed subtype, we determined whether epithelial-to-mesenchymal transition (EMT) had occurred. MATERIALS AND METHODS From the Pan-Pacific TNBC Consortium data set containing TNBC patient samples from four countries, we examined 64 formalin-fixed, paraffin-embedded pairs of matched pre- and post-NST tumor samples. The TNBC subtype was determined using the TNBCtype-IM assay. We analyzed a partial EMT gene expression scoring metric using mRNA data. RESULTS Of the 64 matched pairs, 36 (56%) showed a change in the TNBC subtype after NST. The most frequent change was from BL1 to M subtypes (38%). No tumors changed from M to BL1. The IM signature was positive in 14 (22%) patients before NST and eight (12.5%) patients after NST. The EMT score increased after NST in 28 (78%) of the 36 patients with the changed subtype ( v 39% of the 28 patients without change; P = .002254). CONCLUSION We report, to our knowledge, for the first time that the TNBC molecular subtype and IM signature frequently change after NST. Our results also suggest that EMT is promoted by NST. Our findings may lead to innovative adjuvant therapy strategies in TNBC cases with residual tumor after NST.

Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.

The CARD11-Bcl10-Malt1 (CBM) signalosome controls TAK1 activation to regulate B-cell receptor (BCR)-induced NF-κB activation and B cell biology. The biological function of caspase recruitment domain family member 19 (CARD19), originally identified as a BCL10-interacting CARD protein (BinCARD), is not known. Here we found CARD19 strongly interacted with TAK1 but not BCL10 or other CBM components and prevented TAK1's association with TAB2, thereby inhibiting TAB2-mediated TAK1 ubiquitination and activation and subsequent NF-κB activation. CARD19 was ubiquitously expressed in hematopoietic lineages but its deficiency in mice had no effect on hematopoiesis, including B cell development and humoral immune response. CARD19 deficiency enhanced clonal deletion, receptor editing and anergy of self-reactive B cells, thus reducing autoantibody production in vivo. Mechanistically, CARD19 deficiency led to an increase of BCR/TAK1-mediated NF-κB activation. Activation of NF-κB, such as c-Rel, was responsible for the up-regulation of BCR-induced expression of the transcription factor early growth response genes 2 and 3 (Egr2, Egr3) and the E3 ubiquitin ligases, c-Cbl and Cbl-b, the important inducers of B-cell tolerance in B cells. Further, high-throughput RNA sequencing analysis revealed that CARD19 deficiency did not affect the overall antigen-induced gene expression in naïve B cells but suppressed BCR signaling to increase hyporesponsiveness of self-reactive B cells. Consequently, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus (SLE) and autoimmunity in a B cell-intrinsic manner. Taken together, CARD19 negatively regulates BCR-induced NF-κB activation via blocking TAK1/TAB2 interaction and its deficiency leads to NF-κB-induced expression of Egr2/3 and c-Cbl/Cbl-b in self-reactive B cells, which enhances B-cell tolerance and thus prevents autoimmunity. Disclosures No relevant conflicts of interest to declare.

Ax-101, a small-molecule immunomodulator with a yet-to-be elucidated MoA, enhances lymphocyte proliferation, induces a Th1 profile, increases T-cell cytotoxicity, and improves the M1/M2 ratio. Specific Ax-101 effects include upregulated cell-surface TNFalpha (sTNF) on human T cells and monocytes, increased (>7.5x) T-cell cytotoxicity, upregulation by human PBMC of antitumor cytokines (e.g., IL-2, IFN-gamma, TNFalpha) and downregulation of cytokines that mediate immune evasion (e.g., IL-10), and decreased immune-suppressive myeloid cells. Syngeneic mouse cancer models including bladder (MBT-2), colorectal (CT-26), melanoma (Cloudman), and lymphoma (A20) show the addition of Ax-101 to anti-PD1 to improve outcomes. For example, neither Ax-101 nor anti-PD1 monotherapy had significant effects on mean tumor volume (MTV) in MBT-2 mice, while an Ax-101/anti-PD1 (same doses/regimen) combo resulted in up to 44% reduction in MTV c/w anti-PD1 alone (p < 0.05). Further, 30% (3/10) of MBT-2 mice achieved CR with the combo, whereas no CRs were seen with anti-PD1 alone. In CT-26 mice, 25% (2/8) achieved CR, whereas no CRs occurred with anti-PD1 only; subset analysis of mice exhibiting at least some antitumor response to anti-PD1 alone (6/8) or the Ax-101/anti-PD1 combo (5/8) revealed a 40% CR rate and MTV reduction of up to 75% in combo-treated animals. In Cloudman melanomas, all (5/5) mice receiving Ax-101/anti-PD1 throughout their treatment achieved CR. Importantly, in mice with rapidly growing melanomas refractory to anti-PD1 for 12 d, Ax-101 addition reversed growth, with 4/5 mice achieving CR. Of note, 9 Cloudman mice achieving CR were re-injected with 3x the melanoma cells they initially received and followed with no therapy. Only 1/9 mice developed tumor over 73 d; thus, Ax-101/anti-PD1 may elicit a vaccine-like effect. Anti-PD1 alone and the Ax-101/antiPD1 combo in A20 mice resulted in 23% and 40% lower MTVs c/w control. Ax-101 was previously in Ph 1/2 monotherapy studies in follicular lymphoma (FL) and myeloma (M) (CT.gov IDs: NCT00006466, NCT00007839). Thirty-two patients received 2 µg SQ Ax-101 q2 wkly or wkly for up to 18 months. No Grade 3 or higher toxicities were observed. Five of 14 FL patients showed tumor reduction from 16-64%, while 1/17 M patients showed 50% reduced M-protein; maximum responses occurred between 43 days and over 1 year. Increased PBMC sTNF was seen in all patients tested (8 with FL, 6 with M) (p = 0.0006). Consistent with Ax-101 activity depending on a functional immune system, delayed type hypersensitivity (DTH) testing showed all responders to be DTH+ (5 tumor reduction, 4 SD); 5 anergic (DTH-) patients had SD at best (2 SD, 3 worse). Current Ax-101 clinical development will leverage its beneficial immunostimulatory properties to augment the efficacy of anti-PD1 inhibitors such as pembrolizumab, nivolumab and cemiplimab. Citation Format: Stephan W. Morris, Betsy Fisher, Kent Murphy. Ax-101, an immunostimulatory small molecule, markedly enhances the antitumor activity of PD1 immune checkpoint blockade in multiple preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1720.

Background Triple negative breast cancer (TNBC) is a heterogeneous disease that lacks unifying molecular alterations that can guide therapy decisions. We previously identified distinct molecular subtypes of TNBC (TNBCtype) using gene expression data generated on a microarray platform using frozen tumor specimens. Tumors and cell lines representing the identified subtypes have distinct enrichment in biologically relevant transcripts with differing sensitivity to standard chemotherapies and targeted agents. Since our initial discoveries, RNA-sequencing (RNA-seq) has evolved as a sensitive and quantitative tool to measure transcript abundance. Methods To demonstrate that TNBC subtypes were similar between platforms, we compared gene expression from matched specimens profiled by both microarray and RNA-seq from The Cancer Genome Atlas (TCGA). In the clinical care of patients with TNBC, tumor specimens collected for diagnostic purposes are processed by formalin fixation and paraffin-embedding (FFPE). Thus, for TNBCtype to eventually have broad and practical clinical utility we performed RNA-seq gene expression and molecular classification comparison between fresh-frozen (FF) and FFPE tumor specimens. Results Analysis of TCGA showed consistent subtype calls between 91% of evaluable samples demonstrating conservation of TNBC subtypes across microarray and RNA-seq platforms. We compared RNA-seq performed on 21-paired FF and FFPE TNBC specimens and evaluated genome alignment, transcript coverage, differential transcript enrichment and concordance of TNBC molecular subtype calls. We demonstrate that subtype accuracy between matched FF and FFPE samples increases with sequencing depth and correlation strength to an individual TNBC subtype. Conclusions TNBC subtypes were reliably identified from FFPE samples, with highest accuracy if the samples were less than 4 years old and reproducible subtyping increased with sequencing depth. To reproducibly subtype tumors using gene expression, it is critical to select genes that do not vary due to platform type, tissue processing or RNA isolation method. The majority of differentially expressed transcripts between matched FF and FFPE samples could be attributed to transcripts selected for by RNA enrichment method. While differentially expressed transcripts did not impact TNBC subtyping, they will provide guidance on determining which transcripts to avoid when implementing a gene set size reduction strategy. Trial registration NCT00930930 07/01/2009. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3237-1) contains supplementary material, which is available to authorized users.

Background In patients with triple-negative breast cancer (TNBC), tumor-infiltrating lymphocytes (TILs) have been reported to be associated with improved survival. Lehmann et al. identified 6 molecular subtypes of TNBC [basal-like (BL) 1, BL2, mesenchymal (M), mesenchymal stem like (MSL), immunomodulatory (IM), and luminal androgen receptor (LAR)], and we previously reported that TNBC subtype is a predictor of pathologic complete response (pCR). Recently, the IM gene expression signature has been shown to be indicative of the presence of TILs and has been incorporated into TNBC subtyping as a modifier of the other groups rather than a separate subtype. However, the association between TNBC subtype and the presence of TILs is not known. We hypothesized that the BL2 and LAR subtypes, which have low pCR rates, have low rates of immune infiltration. Inflammatory breast cancer (IBC) is an aggressive cancer that is frequently triple-negative. The association between IBC and the presence of TILs also is not known. In this study, we analyzed the association between TNBC molecular subtype and the IM signature and determined whether the IM signature differed between patients with IBC and non-IBC. Methods We retrospectively analyzed 88 patients with TNBC from the World IBC Consortium dataset for whom IBC status was known (IBC, n=39; non-IBC, n=49) and tumor gene expression data were available. TNBC specimens were classified using the TNBCtype algorithm (Insight Genetics, Inc., TN, USA), which uses a 101-gene signature. For each tumor, the TNBCtype algorithm reports the TNBC molecular subtype (BL1, BL2, M, MSL, or LAR) and the IM status, which is described as positive (IM+) or negative (IM-). Recently, Fisher's exact test was used to analyze differences in subtype distribution between the IM+ and IM- tumors. Results The subtype distribution differed significantly between the IM+ and IM- tumors IM signature in TNBC subtypesSubtypeTotal (n=88)IM+ (n=32)IM- (n=56)BL13015 (50)15 (50)BL2202 (100)M808 (100)MSL3113 (42)18 (58)LAR121 (8)11 (92)Not determined53 (60)2 (40) (p=0.0087). The majority of IM+ cases occurred in the BL1 and MSL subtypes. No IM+ cases were observed in the BL2 or M subtypes, and only 1 was observed in the LAR subtype. IM+ cases occurred at roughly the same frequency in patients with IBC (33%) and non-IBC (37%, p=0.73). Conclusions TNBC molecular subtypes differ in their degree of immune infiltration, and most IM+ TNBCs are of the BL1 and MSL subtypes. Our finding that the proportion of IM+ cases was not different between IBC and non-IBC indicates that TILs are recruited to the tumor microenvironment similarly in IBC and non-IBC tumors. Further, Pietenpol et al recently showed that the MSL signature represents normal stromal cells rather than tumor cells by performing laser-capture microdissection of TNBC specimen. Validation studies are needed to corroborate and further expand upon our findings. Citation Format: Harano K, Wang Y, Lim B, Seitz RS, Morris SW, Bailey DB, Hout DR, Skelton RL, Ring BZ, Masuda H, Rao AUK, Woodward WA, Reuben JM, Ueno NT. Rates of immune infiltration in patients with triple-negative breast cancers by molecular subtype and in patients with inflammatory and non-inflammatory breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-14.

beta 2-integrins are essential for immune system function because they mediate immune cell adhesion and signaling. Consequently, a loss of beta(2)-integrin expression or function causes the immunodeficiency disorders, Leukocyte Adhesion Deficiency (LAD) type I and III. LAD-III is caused by mutations in an important integrin regulator, kindlin-3, but exactly how kindlin-3 regulates leukocyte adhesion has remained incompletely understood. Here we demonstrate that mutation of the kindlin-3 binding site in the beta 2-integrin (TTT/AAA-beta 2-integrin knock-in mouse/KI) abolishes activation of the actin-regulated myocardin related transcription factor A/serum response factor (MRTF-A/SRF) signaling pathway in dendritic cells and MRTF-A/SRF-dependent gene expression. We show that Ras homolog gene family, member A (RhoA) activation and filamentous-actin (F-actin) polymerization is abolished in murine TTT/AAA-beta 2-integrin KI dendritic cells, which leads to a failure of MRTF-A to localize to the cell nucleus to coactivate genes together with SRF. In addition, we show that dendritic cell gene expression, adhesion and integrin-mediated traction forces on ligand coated surfaces is dependent on the MRTF-A/SRF signaling pathway. The participation of beta 2-integrin and kindlin-3-mediated cell adhesion in the regulation of the ubiquitous MRTF-A/SRF signaling pathway in immune cells may help explain the role of beta 2-integrin and kindlin-3 in integrin-mediated gene regulation and immune system function.

International audience; In patients with triple-negative breast cancer (TNBC), tumor-infiltrating lymphocytes (TILs) are associated with improved survival. Lehmann et al. identified 4 molecular subtypes of TNBC [basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor (LAR)], and an immunomodulatory (IM) gene expression signature indicates the presence of TILs and modifies these subtypes. The association between TNBC subtype and TILs is not known. Also, the association between inflammatory breast cancer (IBC) and the presence of TILs is not known. Therefore, we studied the IM subtype distribution among different TNBC subtypes. We retrospectively analyzed patients with TNBC from the World IBC Consortium dataset. The molecular subtype and the IM signature [positive (IM+) or negative (IM-)] were analyzed. Fisher's exact test was used to analyze the distribution of positivity for the IM signature according to the TNBC molecular subtype and IBC status. There were 88 patients with TNBC in the dataset, and among them 39 patients (44%) had IBC and 49 (56%) had non-IBC. The frequency of IM+ cases differed by TNBC subtype (p = 0.001). The frequency of IM+ cases by subtype was as follows: BL1, 48% (14/29); BL2, 30% (3/10); LAR, 18% (3/17); and M, 0% (0/21) (in 11 patients, the subtype could not be determined). The frequency of IM+ cases did not differ between patients with IBC and non-IBC (23% and 33%, respectively; p = 0.35). In conclusion, the IM signature representing the underlying molecular correlate of TILs in the tumor may differ by TNBC subtype but not by IBC status.

Local ablative therapy with stereotactic ablative radiotherapy may improve survival in oncogene‐addicted lung cancer patients with extracranial oligometastatic disease treated with targeted therapies. There is limited data on the use of radiofrequency ablation (RFA) in this same setting. We present a case of an anaplastic lymphoma kinase (ALK)‐positive lung cancer patient with hepatic oligometastatic progression who was successfully treated with both stereotactic ablative radiation and RFA while continuing with an ALK inhibitor.

Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

Ruxolitinib is the only JAK2 inhibitor approved for myelofibrosis (MF). Although JAK2 inhibitor therapy improves splenomegaly and systemic symptoms, it does not significantly reduce the MF clone or alter the natural history and survival in most patients. The novel small molecule OHM-581 not only inhibits JAK2, but also BET (bromodomain and extra terminal) proteins such as BRD4. Dual JAK2 and BET inhibition using other molecules has shown superior efficacy in murine MF models compared with JAK2 inhibition alone including amelioration of fibrosis, synergistic induction of apoptosis of primary patient, post-MF secondary acute myeloid leukemia (sAML) blasts, and significant improvement of the survival of mice engrafted with human sAML cells. OHM-581 is being developed for the treatment of MF and other hematologic malignancies to leverage these advantages of dual JAK2-BET inhibition. Results: In vitro biochemical assays show OHM-581 to inhibit BRD4 BD1 and BD2 with IC50s of 189 and 116 nM, respectively, and JAK2 with an IC50 of 7.1 nM (JAK1=244 nM; JAK3=76 nM). No significant hERG liability was seen. OHM-581 displays significant anti-proliferative activity against multiple liquid cancer cell lines. Notably, the GI50 values for MV4-11 (AML, MLL fusion+), HEL 92.1.7 (AML, JAK2V617F+) and UKE-1 (AML, JAK2V617F+) are 32, 87 and 140 nM, respectively. Consistent with its mechanism of action, OHM-581 robustly down-regulates cMYC expression (cMYC mRNA down-regulation IC50=18.6 nM compared with 41.4 nM for BET inhibitor JQ1) and JAK2 signaling. Moreover, the compound triggers apoptosis in MV4-11 cells (~7-fold increase in Caspase 3/7 activity at 24-h). OHM-581 does not reflect an efflux liability, the efflux ratio being nearly 1.8. OHM-581 is soluble in PBS pH7.4 at 26 µM and metabolically stable with nearly 51%, 67% and 88% remaining in mouse, rat and human liver microsomes, respectively, after 30 min. OHM-581 exhibits an oral bioavailability of ~40%. As compared to the small molecule fedratinib, which is also being developed for MF, OHM-581 does not inhibit thiamine uptake. Dose proportionality with a greater than proportional increase in both Cmax and AUC at doses up to 60 mg/kg, and a saturation of Cmax at doses of 60 mg/kg and higher, are observed for OHM-581. In vivo efficacy studies using an MV4-11 xenograft model demonstrate robust tumor inhibition (~75-90%) following doses of 30 and 60 mg/kg po BID for 20 days. Conclusion: OHM-581 is a novel dual inhibitor of JAK2 and BET that has potential as a therapeutic agent for MF and other hematologic malignancies. In addition to IND-enabling studies, current OHM-581 development activities are focused on further proof-of-concept analyses using preclinical models of MF and other myeloproliferative neoplasms, with results to be reported. Citation Format: Ram S. Upadhayaya, Raghava Reddy Kethiri, Stephan W. Morris, H. Michael Shepard, Samuel R. Saks, Michael J. Weickert. OHM-581, a dual JAK2-BET inhibitor for the treatment of myelofibrosis and other hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3067.

Introduction: Treatment of TNBC has been challenging due to the absence of well-characterized molecular targets and the heterogeneity of the disease. Using TNBC gene expression (GE) microarray profiles, the Pietenpol group molecularly binned the malignancy into six distinct subtypes: two basal-like [BL1, BL2], two mesenchymal-like [M, MSL], an immunomodulatory [IM], and a luminal subtype expressing androgen receptor [LAR]). Importantly, subtype-specific TNBC cell lines exhibit different sensitivities to various targeted and conventional chemotherapies currently employed or under investigation for the treatment of TNBC (1). Background: The original TNBCtype algorithm was generated from a meta-analysis of existing GE from tumor tissue and clustering the data into the six subtypes listed above and a seventh "unclassified" subtype (1). To transition the test into the clinic, we have modified the method of classification by both reducing the number of signature genes and normalizing the data against a reference set of endogenous expressed genes. Methods: Gene set enrichment followed by shrunken centroid analysis were used for feature reduction, resulting in 258 genes used for model building. The IM class was excluded from the feature reduction analysis as it likely represents presence of immune infiltrates rather than a distinct tumor class. Linear regression, targeted minimum loss based estimation, random forest, and elastic-net regularized linear models were employed, with the latter giving the best fit with the least number of required genes. Models were created to identify each class individually or together using a multiclass model. Coefficient and cutoffs were established on a Robust Multichip Average (RMA) normalized TNBC training data set consisting of 14 cohorts (N=386) and then applied to a seven cohort validation data set (N=201). A reference gene set was chosen using three of the training cohorts with the criteria of low intra- and inter-cohort variation, as well as overall low coefficient of variation, low probe-to-probe variability, expression greater than the cohort mean, and functional diversity. New cutoffs were determined using these same three cohorts, and normalization with the reference genes was tested using an additional two cohorts from the training data. Results: In the RMA normalized validation data set all models showed significant classification, (Fisher exact test, P Conclusions: These results indicate that information conveyed in the initial clustering algorithm can be similarly obtained in a single patient sample using a reduced gene set normalized to internal controls. Future work will determine the biologic and clinical utility of this assay for patient management. Citation Format: Rob S Seitz, David R Hout, Stephan W Morris, Rebecca B Smith, Brian D Lehmann, Xi Chen, Jennifer A Pietenpol, Brian Z Ring. Creation of a robust algorithm utilizing minimal gene sets normalized against a reference gene set to identify triple-negative breast cancer (TNBC) subtypes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-04.

A20, a tumor suppressor in several types of lymphomas, has been suggested to be an nuclear factor kappa B (NF-κB) target gene; conversely, the deubiquitylation activity of A20 is required for inhibition of Bcl10-mediated activation of NF-κB. BCL10, which is activated in a recurrent chromosomal translocation that causes human mucosa-associated lymphoid tissue lymphomas, is known to be essential for NF-κB activation in B cells. We report here that Bcl10 upregulates endogenous A20 gene expression in B lymphocytes upon B-cell receptor engagement of anti-IgM. Transient transfection assays in HEK 293 cells indicate that Bcl10 can activate the A20 promoter, which contains NF-κB-binding sites. We also construct a theoretical structure of mouse Bcl10 and analyze the structure by molecular modeling and molecular dynamics simulation. Lastly, we found that marginal zone B cells from BCL10-transgenic mice proliferate more readily than wild-type B cells, whereas, surprisingly, the transgenic follicular B cells from these mice proliferate comparably to wild-type cells. Collectively, our results indicate that Bcl10 is an essential regulator of A20 gene expression and B-cell proliferation mediated by B-cell receptor signaling.

The common food additive carrageenan is a known activator of inflammation in mammalian tissues and stimulates both the canonical and noncanonical pathways of NF-κB activation. Exposure to low concentrations of carrageenan (10 μg/mL in the water supply) has produced glucose intolerance, insulin resistance, and impaired insulin signaling in C57BL/6 mice. B-cell leukemia/lymphoma 10 (Bcl10) is a mediator of inflammatory signals from Toll-like receptor (TLR) 4 in myeloid and epithelial cells. Since the TLR4 signaling pathway is activated in diabetes and by carrageenan, we addressed systemic and intestinal inflammatory responses following carrageenan exposure in Bcl10 wild type, heterozygous, and null mice. Fecal calprotectin and circulating keratinocyte chemokine (KC), nuclear RelA and RelB, phospho(Thr559)-NF-κB-inducing kinase (NIK), and phospho(Ser36)-IκBαin the colonic epithelial cells were significantly less (P<0.001) in the carrageenan-treated Bcl10 null mice than in controls. IL-10-deficient mice exposed to carrageenan in a germ-free environment showed an increase in activation of the canonical pathway of NF-κB (RelA) activation, but without increase in RelB or phospho-Bcl10, and exogenous IL-10 inhibited only the canonical pathway of NF-κB activation in cultured colonic cells. These findings demonstrate a Bcl10 requirement for maximum development of carrageenan-induced inflammation and lack of complete suppression by IL-10 of carrageenan-induced inflammation.

In the HAX1/HtrA2-OMI/PARL (HOP) mitochondrial protein complex, anti-apoptotic signals are generated by cleavage and activation of the serine protease HtrA2/OMI by the rhomboid protease PARL upon recruitment of both proteases to inner mitochondrial membrane protein HAX1 (HS1-associated protein X-1). Here we report the negative regulation of the HOP complex by human leukemia-associated myeloid leukemia factor 1 (MLF1). We demonstrate that MLF1 physically and functionally associates with HAX1 and HtrA2. Increased interaction of MLF1 with HAX1 and HtrA2 displaces HtrA2 from the HOP complex and inhibits HtrA2 cleavage and activation, resulting in the apoptotic cell death. Conversely, over-expressed HAX1 neutralizes MLF1's effect and inhibits MLF1-induced apoptosis. Importantly, Mlf1 deletion reverses B- and T-cell lymphopenia and significantly ameliorates the progressive striatal and cerebellar neurodegeneration observed in Hax1-/- mice, with a doubling of the lifespan of Mlf1-/-/Hax1-/- animals compared to Hax1-/- animals. Collectively, these data indicate that MLF1 serves as a proapoptotic antagonist that interacts with the HOP mitochondrial complex to modulate cell survival.

Recently, a gene expression algorithm, TNBCtype, was developed that can divide triple-negative breast cancer (TNBC) into molecularly-defined subtypes. The algorithm has potential to provide predictive value for TNBC subtype-specific response to various treatments. TNBCtype used in a retrospective analysis of neoadjuvant clinical trial data of TNBC patients demonstrated that TNBC subtype and pathological complete response to neoadjuvant chemotherapy were significantly associated. Herein we describe an expression algorithm reduced to 101 genes with the power to subtype TNBC tumors similar to the original 2188-gene expression algorithm and predict patient outcomes.The new classification model was built using the same expression data sets used for the original TNBCtype algorithm. Gene set enrichment followed by shrunken centroid analysis were used for feature reduction, then elastic-net regularized linear modeling was used to identify genes for a centroid model classifying all subtypes, comprised of 101 genes. The predictive capability of both this new "lean" algorithm and the original 2188-gene model were applied to an independent clinical trial cohort of 139 TNBC patients treated initially with neoadjuvant doxorubicin/cyclophosphamide and then randomized to receive either paclitaxel or ixabepilone to determine association of pathologic complete response within the subtypes.The new 101-gene expression model reproduced the classification provided by the 2188-gene algorithm and was highly concordant in the same set of seven TNBC cohorts used to generate the TNBCtype algorithm (87%), as well as in the independent clinical trial cohort (88%), when cases with significant correlations to multiple subtypes were excluded. Clinical responses to both neoadjuvant treatment arms, found BL2 to be significantly associated with poor response (Odds Ratio (OR) =0.12, p=0.03 for the 2188-gene model; OR = 0.23, p0.03 for the 101-gene model). Additionally, while the BL1 subtype trended towards significance in the 2188-gene model (OR = 1.91, p = 0.14), the 101-gene model demonstrated significant association with improved response in patients with the BL1 subtype (OR = 3.59, p = 0.02).These results demonstrate that a model using small gene sets can recapitulate the TNBC subtypes identified by the original 2188-gene model and in the case of standard chemotherapy, the ability to predict therapeutic response.

Background Recently, a gene expression algorithm, TNBCtype, was developed that can divide triple-negative breast cancer (TNBC) into molecularly-defined subtypes. The algorithm has potential to provide predictive value for TNBC subtype-specific response to various treatments. TNBCtype used in a retrospective analysis of neoadjuvant clinical trial data of TNBC patients demonstrated that TNBC subtype and pathological complete response to neoadjuvant chemotherapy were significantly associated. Herein we describe an expression algorithm reduced to 101 genes with the power to subtype TNBC tumors similar to the original 2188-gene expression algorithm and predict patient outcomes. Methods The new classification model was built using the same expression data sets used for the original TNBCtype algorithm. Gene set enrichment followed by shrunken centroid analysis were used for feature reduction, then elastic-net regularized linear modeling was used to identify genes for a centroid model classifying all subtypes, comprised of 101 genes. The predictive capability of both this new “lean” algorithm and the original 2188-gene model were applied to an independent clinical trial cohort of 139 TNBC patients treated initially with neoadjuvant doxorubicin/cyclophosphamide and then randomized to receive either paclitaxel or ixabepilone to determine association of pathologic complete response within the subtypes. Results The new 101-gene expression model reproduced the classification provided by the 2188-gene algorithm and was highly concordant in the same set of seven TNBC cohorts used to generate the TNBCtype algorithm (87 %), as well as in the independent clinical trial cohort (88 %), when cases with significant correlations to multiple subtypes were excluded. Clinical responses to both neoadjuvant treatment arms, found BL2 to be significantly associated with poor response (Odds Ratio (OR) =0.12, p =0.03 for the 2188-gene model; OR = 0.23, p

The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness–induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix–induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling–mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism.

We report the synthesis of a pyrimidinone library that targets anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase. This library was generated in three steps from a versatile commercially available starting material. Some compounds within this library showed single digit micromolar inhibition of ALK in vitro, while showing minimal inhibition of other homologous insulin receptor family kinases including the human insulin receptor kinase (IRK), at the highest concentrations investigated. We also present initial ALK structure–activity relationships for this library.

12069Background: Lehmann et al have identified 4 molecular subtypes of TNBC [basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor (LAR)] and an IM gene expression signature. Our g...

BCL10, required for nuclear factor κB (NF-κB) activation during antigen-driven lymphocyte responses, is aberrantly expressed in mucosa-associated lymphoid tissue-type marginal zone (MZ) lymphomas because of chromosomal translocations. Eμ-driven human BCL10 transgenic (Tg) mice, which we created and characterize here, had expanded populations of MZ B cells and reduced follicular and B1a cells. Splenic B cells from Tg mice exhibited constitutive activation of both canonical and noncanonical NF-κB signaling pathways is associated with increased expression of NF-κB target genes. These genes included Tnfsf13b, which encodes the B-cell activating factor (BAFF). In addition, levels of BAFF were significantly increased in sera from Tg mice. MZ B cells of Tg mice exhibited reduced turnover in vivo and enhanced survival in vitro, indicative of lymphoaccumulation rather than lymphoproliferation as the cause of MZ expansion. In vivo antibody responses to both T-independent, and especially T-dependent, antigens were significantly reduced in Tg mice. Mortality was accelerated in Tg animals, and some mice older than 8 months had histologic and molecular findings indicative of clonal splenic MZ lymphoma. These results suggest that, in addition to constitutive activation of BCL10 in MZ B cells, other genetic factors or environmental influences are required for short latency oncogenic transformation.

RNA-binding motif protein 15 (RBM15) is involved in the RBM15-megakaryoblastic leukemia 1 fusion in acute megakaryoblastic leukemia. Although Rbm15 has been reported to be required for B-cell differentiation and to inhibit myeloid and megakaryocytic expansion, it is not clear what the normal functions of Rbm15 are in the regulation of hematopoietic stem cell (HSC) and megakaryocyte development. In this study, we report that Rbm15 may function in part through regulation of expression of the proto-oncogene c-Myc. Similar to c-Myc knockout (c-Myc-KO) mice, long-term (LT) HSCs are significantly increased in Rbm15-KO mice due to an apparent LT-HSC to short-term HSC differentiation defect associated with abnormal HSC-niche interactions caused by increased N-cadherin and β1 integrin expression on mutant HSCs. Both serial transplantation and competitive reconstitution capabilities of Rbm15-KO LT-HSCs are greatly compromised. Rbm15-KO and c-Myc-KO mice also share related abnormalities in megakaryocyte development, with mutant progenitors producing increased, abnormally small low-ploidy megakaryocytes. Consistent with a possible functional interplay between Rbm15 and c-Myc, the megakaryocyte increase in Rbm15-KO mice could be partially reversed by ectopic c-Myc. Thus, Rbm15 appears to be required for normal HSC-niche interactions, for the ability of HSCs to contribute normally to adult hematopoiesis, and for normal megakaryocyte development; these effects of Rbm15 on hematopoiesis may be mediated at least in part by c-Myc.

It has been found that c-Myc protein plays a critical role in controlling self-renewal versus differentiation in hematopoietic stem cells. We report that c-Myc also controls the fate of megakaryocyte-erythrocyte progenitors through regulating the differentiation of erythroid and megakaryocytic progenitors. In addition to the significant reduction of granulocytes/macrophages and B and T lymphocytes because of the reduction of their corresponding progenitors, we found significantly increased numbers of megakaryocytic progenitors and mature megakaryocytes in bone marrow and spleens of c-Myc-knockout (c-Myc−/−) mice. Differentiation of erythrocytes was blocked at the erythroid progenitor stage. This increased megakaryocytopoiesis is a cell-intrinsic defect of c-Myc-mutant hematopoietic stem cells, as shown by transplantation studies. Furthermore, we found that c-Myc is required for polyploidy formation but not for cytoplasmic maturation of megakaryocytes. Megakaryocytes from c-Myc−/− mice are significantly smaller in size and lower in ploidy than those of control mice; however, because of the dramatic increase in megakaryocyte number, although fewer platelets are produced by each megakaryocyte, a greater than 3-fold increase in platelet number was consistently observed in c-Myc−/− mice. Thus, c-Myc−/− mice develop a syndrome of severe thrombocytosis-anemia-leukopenia because of significant increases in megakaryocytopoiesis and concomitant blockage of erythrocyte differentiation and reductions in myelolymphopoiesis.

We report the design and synthesis of an insulin receptor kinase family-targeted inhibitor template using the inhibitor conformation observed in an IGF1R/inhibitor co-crystal complex by application of a novel molecular design approach that we have recently published. The synthesis of the template involves a one pot Opatz cyclization reaction that provides a versatile indole ester in good yields. We also developed the required chemistry to elaborate this template with additional substituents and have used this chemistry to prepare some initial compounds that show selective inhibition of anaplastic lymphoma kinase (ALK).

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

We report the design and highly enantioselective synthesis of a potent analogue of the spliceosome inhibitor FR901464, based on a non-natural product scaffold. The design of this compound was facilitated by a pharmacophore hypothesis that assumed key interaction types that are common to FR901464 and an otherwise unrelated natural product (pladienolide). The synthesis allows for the preparation of numerous novel analogues. We present results on the in vitro activity for this compound against several tumor cell lines.

Previous studies have demonstrated that Bcl10 (B-cell leukemia/lymphoma 10) is essential for T cell receptor-mediated NF-κB activation and subsequent proliferation and interleukin 2 (IL2) production. However, here we demonstrate that, contrary to expectations, Bcl10 is differentially required for T cell activation, including for both proliferation and cytokine production. When CD4+ and CD8+ T cells were divided based on expression levels of CD44, which distinguishes naive cells (CD44lo) versus those that are antigen-experienced (CD44hi), IL2 production by and proliferation of CD4+CD44lo naive cells and both subpopulations of CD8+ T cells were clearly Bcl10-dependent, whereas these same functional properties of CD4+CD44hi T cells occurred largely independent of Bcl10. As with the other subpopulations of T cells, CD4+CD44hi T cells did not activate the NF-κB pathway in the absence of Bcl10; nevertheless, these CD4+CD44hi antigen-experienced T cells efficiently secreted IL2 after T cell receptor stimulation. Strikingly, therefore, T cell receptor-mediated IL2 production in these cells is NF-κB-independent. Our studies suggest that antigen-experienced CD4+ T cells differ from their naive counterparts and from CD8+ T cells in their ability to achieve activation independent of the Bcl10/NF-κB pathway.

Activating NK cell receptors transduce signals through ITAM-containing adaptors, including FcRγ and DAP12. Although the caspase recruitment domain (CARD)9-Bcl10 complex is essential for FcRγ/DAP12-mediated NF-κB activation in myeloid cells, its involvement in NK cell receptor signaling is unknown. Herein we show that the deficiency of CARMA1 or Bcl10, but not CARD9, resulted in severe impairment of cytokine/chemokine production mediated by activating NK cell receptors due to a selective defect in NF-κB activation, whereas cytotoxicity mediated by the same receptors did not require CARMA1-Bcl10-mediated signaling. IκB kinase (IKK) activation by direct protein kinase C (PKC) stimulation with PMA plus ionomycin (P/I) was abrogated in CARMA1-deficient NK cells, similar to T and B lymphocytes, whereas CARD9-deficient dendritic cells (DCs) exhibited normal P/I-induced IKK activation. Surprisingly, CARMA1 deficiency also abrogated P/I-induced IKK activation in DCs, indicating that CARMA1 is essential for PKC-mediated NF-κB activation in all cell types, although the PKC-CARMA1 axis is not used downstream of myeloid ITAM receptors. Consistently, PKC inhibition abrogated ITAM receptor-mediated activation only in NK cells but not in DCs, suggesting PKC-CARMA1-independent, CARD9-dependent ITAM receptor signaling in myeloid cells. Conversely, the overexpression of CARD9 in CARMA1-deficient cells failed to restore the PKC-mediated NF-κB activation. Thus, NF-κB activation signaling through ITAM receptors is regulated by a cell type-specific mechanism depending on the usage of adaptors CARMA1 and CARD9, which determines the PKC dependence of the signaling.

Nuclear interaction partner of ALK (NIPA) is an F-box-containing protein that defines a nuclear skp1 cullin F-box (SCF)-type ubiquitin E3 ligase (SCFNIPA) implicated in the regulation of mitotic entry. The SCFNIPA complex targets nuclear cyclin B1 for ubiquitination in interphase, whereas phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. In addition to the recently described serine residue 354, we specify 2 new residues, Ser-359 and Ser-395, implicated in the phosphorylation process at G2/M within this region. Moreover, we found cyclin B1/Cdk1 to phosphorylate NIPA at Ser-395 in mitosis. Mutation of both Ser-359 and Ser-395 impaired effective inactivation of the SCFNIPA complex, resulting in reduced levels of mitotic cyclin B1. These data are compatible with a process of sequential NIPA phosphorylation where cyclin B1/Cdk1 amplifies phosphorylation of NIPA once an initial phosphorylation event has dissociated the SCFNIPA complex. Thus, cyclin B1/Cdk1 may contribute to the regulation of its own abundance in early mitosis.

RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, we must understand the normal functions of RBM15 and MKL. Here, we show a role for Rbm15 in myelopoiesis. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. We show that Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJkappa, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJkappa.

The adaptor protein B cell lymphoma 10 (Bcl10) plays an essential role in the functions of the AgRs in T and B cells. In this study, we report that Bcl10 also plays an important role in mast cells. Bcl10 is expressed in mast cells. Although Bcl10-deficient mast cells undergo normal development, we demonstrate that Bcl10 is essential for specific functions of FcεR. Although Bcl10-deficient mast cells have normal de novo synthesis and release of the lipid mediator arachidonic acid, the mutant cells possess impaired FcεR-mediated degranulation, indicated by decreased serotonin release, and impaired cytokine production, measured by release of IL-6. In addition, Bcl10-deficient mice display impaired IgE-mediated passive cutaneous anaphylaxis. Moreover, although Bcl10-deficient mast cells have normal FcεR-mediated Ca2+ flux, activation of PI3K, and activation of the three types of MAPKs (ERKs, JNK, and p38), the mutant cells have markedly diminished FcεR-mediated activation of NF-κB and decreased activation of AP-1. Thus, Bcl10 is essential for FcεR-induced activation of AP-1, NF-κB, degranulation, and cytokine production in mast cells.

Background The anaplastic lymphoma kinase (ALK) gene encodes a receptor tyrosine kinase, which was first identified as the fusion partner of the nucleophosmin (NPM1) gene in the recurrent t(2;5)(p23;q35) found in a subset of anaplastic large cell lymphoma (ALCL). Several distinct, non-NPM1, ALK fusions have subsequently been described in lymphomas and other tumor types. All of these fusions result in the constitutive expression and activation of ALK and ALK signaling pathways, ultimately leading to the malignant phenotype. Case report A non-NPM1 fusion partner of ALK was identified in a 32-year-old Caucasian male ALCL patient whose disease was refractory to standard chemotherapy and autologous stem cell transplantation, and exhibited a poor response to a first-generation ALK inhibitor. Non-allele-specific ALK RT-qPCR revealed ALK overexpression and 5′ RACE PCR revealed that the patient’s lymphoma expressed a TRAF1-ALK fusion. Conclusions We report the case of an ALCL patient whose tumor harbored the newly recognized TRAF1-ALK fusion and describe the clinical outcome after treatment with an ALK inhibitor. The short survival of our patient may reflect a propensity toward aggressive behavior in lymphomas that express this ALK fusion.

Myeloid leukemia factor 1 (MLF1) was involved in t(3;5) chromosomal rearrangement and aberrantly expressed in myelodysplastic syndromes/acute myeloid leukemia patients. Ex vivo experiments showed that the lymphocytes from the Mlf1-deficient mice were more resistant to apoptotic stimulations than the wild-type cells. Furthermore, the ectopically expressed MLF1 induced apoptosis in the cell models. These findings revealed that MLF1 was required for the cells to respond to the apoptotic stimulations. Ex vivo experiments also demonstrated that cytokine withdrawal significantly up-regulated Mlf1's expression and promoted its association with B cell lymphoma-extra large (Bcl-XL) in the lymphocytes, at the same time reduced the association of Bax with Bcl-XL The same effects were also observed in the cells that over-expressed MLF1. However, these effects were observed in Mlf1 null lymphocytes as well as the cells over-expressing Bcl-XL. In addition, MLF1's proapoptosis could be completely prevented by co-expression of Bcl-XL and significantly attenuated in Bax/Bak double null cells. These data, taken together, strongly suggested that in response to the stresses, up-regulated Mlf1 promoted its association with Bcl-XL and reduced the available Bcl-XL for associating with Bax, which resulted in releasing Bax from the Bcl-XL and apoptosis in turn. Lastly, we showed that MLF1 was negatively regulated by 14-3-3 and revealed that 14-3-3 bound to MLF1 and physically blocked MLF1's Bcl-2 homology domain 3 (BH3) as well as Bcl-XL from associating with MLF1. Our findings suggested that ectopically expressed MLF1 could be responsible for the pathological apoptosis in early myelodysplastic syndrome (MDS) patients.

Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): A comparison with NPMc+ AML

SummaryThe regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-box subunit of the SCF specifically recruiting a given substrate to the SCF core. Here we identify NIPA (nuclear interaction partner of ALK) as a human F-box-containing protein that defines an SCF-type E3 ligase (SCFNIPA) controlling mitotic entry. Assembly of this SCF complex is regulated by cell-cycle-dependent phosphorylation of NIPA, which restricts substrate ubiquitination activity to interphase. We show nuclear cyclin B1 to be a substrate of SCFNIPA. Inactivation of NIPA by RNAi results in nuclear accumulation of cyclin B1 in interphase, activation of cyclin B1-Cdk1 kinase activity, and premature mitotic entry. Thus, SCFNIPA-based ubiquitination may regulate S-phase completion and mitotic entry in the mammalian cell cycle.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, the normal role of which remains to be completely elucidated. Although work carried out in mammals suggests a function in neural development, results from studies in Drosophila indicate an additional role in visceral muscle differentiation. The aberrant expression of full-length ALK receptor proteins has been reported in neuroblastomas and glioblastomas, while the occurrence of ALK fusion proteins in anaplastic large cell lymphoma (ALCL) has resulted in the identification of the new tumor entity, ALK-positive ALCL. ALK represents one of few examples of a receptor tyrosine kinase implicated in oncogenesis in both haematopoietic and non-haematopoietic tumors, given that ALK fusions also occur in the mesenchymal tumor known as inflammatory myofibroblastic tumor (IMT). The study of ALK fusion proteins, besides demonstrating their importance in tumor development, has also raised the possibility of new therapeutic treatments for patients with ALK-positive malignancies.

Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

The normal functions of full-length anaplastic lymphoma kinase (ALK) remain to be completely elucidated. Although considered to be important in neural development, recent studies in Drosophila also highlight a role for ALK in gut muscle differentiation. Indeed, the Drosophila model offers a future arena for the study of ALK, its ligands and signalling cascades. The discovery of activated fusion forms of the ALK tyrosine kinase in anaplastic large cell lymphoma (ALCL) has dramatically improved our understanding of the pathogenesis of these lymphomas and enhanced the pathological diagnosis of this subtype of non-Hodgkin's lymphoma (NHL). Likewise, the realisation that a high percentage of inflammatory myofibroblastic tumours express activated-ALK fusion proteins has clarified the causation of these mesenchymal neoplasms and provided for their easier discrimination from other mesenchymal-derived inflammatory myofibroblastic tumour (IMT) mimics. Recent reports of ALK expression in a range of carcinoma-derived cell lines together with its apparent role as a receptor for PTN and MK, both of which have been implicated in tumourigenesis, raise the possibility that ALK-mediated signalling could play a role in the development and/or progression of a number of common solid tumours. The therapeutic targeting of ALK may prove to have efficacy in the treatment of many of these neoplasms.

Anaplastic large-cell lymphomas (ALCLs) are lymphomas of T or null phenotype often associated with a chromosomal translocation, t(2;5)(p23;q35). This translocation leads to the expression of a hybrid protein consisting of the N-terminal portion of nucleophosmin (NPM) and the intracellular domain of the anaplastic lymphoma kinase (ALK). NPM-ALK possesses a constitutive tyrosine kinase activity responsible for its oncogenic property through activation of downstream effectors such as phospholipase C gamma (PLC-gamma) and the type IA phosphoinositide 3-kinase. Here, we show that the Src-kinases, particularly pp60(c-src), associate with and are activated by NPM-ALK expression in various cells, and in cell lines established from patients with ALCL. The kinase activity and the tyrosine 418 of NPM-ALK are required for its association with Src-kinases. Y418F mutation of NPM-ALK impaired its association with Src-kinases and strongly reduced the proliferation rate of Ba/F3 cells. In agreement, Src-kinase inhibitors or pp60(c-src) siRNA significantly decreased the proliferation rate of NPM-ALK-positive ALCL cell lines. Moreover, using active or inactive forms of pp60(c-src) and NPM-ALK, we provide evidence that NPM-ALK is a potential substrate of pp60(c-src). Overall, our data place Src-kinases as new important downstream effectors of NPM-ALK and as attractive potential therapeutic targets for new ALCL treatment.

PLCgamma2 plays a critical role in B cell receptor (BCR) signaling and its targeted deletion results in defective B cell development and function. Here, we show that PLCgamma2 deficiency specifically blocks B cell maturation at the transitional type 2 (T2) to follicular (FO) B cell transition and the PLCgamma2 pathway regulates survival of B cells. BCR-induced apoptosis is dramatically enhanced in all subsets of splenic PLCgamma2-deficient B cells, especially in T2 and FO B cell subpopulations. We also find that all splenic PLCgamma2-deficient B cell subpopulations express abnormally low levels of Bcl-2 protein. In addition, PLCgamma2 deficiency disrupts BCR-mediated induction of A1 expression. Enforced expression of Bcl-2 prevents BCR-induced apoptosis in all splenic PLCgamma2-deficient B cell subpopulations and partially restores the numbers of PLCgamma2-deficient FO B cells. In contrast to Bcl-2, enforced expression of A1 preferentially prevents BCR-induced apoptosis in PLCgamma2-deficient FO B cells and partially restores the numbers of these B cells. Therefore, the PLCgamma2 pathway provides a survival signal via regulation of Bcl-2 in all splenic B cell subpopulations and via additional induction of A1 in mature FO B cells.

Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that we found strongly activated serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, we assessed its role in serum induction of c-fos and other SRE-regulated genes with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). We found that DN-MKL1 and RNAi specifically blocked SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi required the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduced the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, was nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia had a markedly increased ability to activate SRE reporter genes, suggesting that its activation of SRF target genes may contribute to leukemogenesis.

Bcl10 is an intracellular protein essential for nuclear factor (NF)-kappaB activation after lymphocyte antigen receptor stimulation. Using knockout mice, we show that absence of Bcl10 impeded conversion from transitional type 2 to mature follicular B cells and caused substantial decreases in marginal zone and B1 B cells. Bcl10-deficient B cells showed no excessive apoptosis. However, both Bcl10-deficient follicular and marginal zone B cells failed to proliferate normally, although Bcl10-deficient marginal zone B cells uniquely failed to activate NF-kappaB efficiently after stimulation with lipopolysaccharide. Bcl10-deficient marginal zone B cells did not capture antigens, and Bcl10-deficient (Bcl10-/-) mice failed to initiate humoral responses, leading to an inability to clear blood-borne bacteria. Thus, Bcl10 is essential for the development of all mature B cell subsets.