Loss of FBXO31-mediated degradation of DUSP6 dysregulates ERK and PI3K-AKT signaling and promotes prostate tumorigenesis
FBXO31 is the substrate receptor of one of many CUL1-RING ubiquitin ligase (CRL1) complexes. Here, we show that low FBXO31 mRNA levels are associated with high pre-operative prostate-specific antigen (PSA) levels and Gleason grade in human prostate cancer. Mechanistically, the ubiquitin ligase CRL1FBXO31 promotes the ubiquitylation-mediated degradation of DUSP6, a dual specificity phosphatase that dephosphorylates and inactivates the extracellular-signal-regulated kinase-1 and -2 (ERK1/2). Depletion of FBXO31 stabilizes DUSP6, suppresses ERK signaling, and activates the PI3K-AKT signaling cascade. Moreover, deletion of FBXO31 promotes tumor development in a mouse orthotopic model of prostate cancer. Treatment with BCI, a small molecule inhibitor of DUSP6, suppresses AKT activation and prevents tumor formation, suggesting that the FBXO31 tumor suppressor activity is dependent on DUSP6. Taken together, our studies highlight the relevance of the FBXO31-DUSP6 axis in the regulation of ERK- and PI3K-AKT-mediated signaling pathways, as well as its therapeutic potential in prostate cancer.
Improving prognostic assignment in older adults with multiple myeloma using acquired genetic features, clonal hemopoiesis and telomere length
CRL4AMBRA1 is a master regulator of D-type cyclins
D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, byÂ combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces theÂ accumulation of D-type cyclins andÂ retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous systemÂ that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.
The molecular make up of smoldering myeloma highlights the evolutionary pathways leading to multiple myeloma
Smoldering myeloma (SMM) is associated with a high-risk of progression to myeloma (MM). We report the results of aÂ study of 82 patients with both targeted sequencing that included a capture of the immunoglobulin and MYC regions. By comparing these results to newly diagnosed myeloma (MM) we show fewer NRAS and FAM46C mutations together with fewer adverse translocations, del(1p), del(14q), del(16q), and del(17p) in SMM consistent with their role as drivers of the transition to MM. KRAS mutations are associated with a shorter time to progression (HR 3.5 (1.5-8.1), pâ€‰=â€‰0.001). In an analysis of change in clonal structure over time we studied 53 samples from nine patients at multiple time points. Branching evolutionary patterns, novel mutations, biallelic hits in crucial tumour suppressor genes, and segmental copy number changes are key mechanisms underlying the transition to MM, which can precede progression and be used to guide early intervention strategies.
CSF plasmablasts differentiate MS from other neurologic disorders [Letter]
Multiparametric flow cytometry (FC) of CSF allows one to easily estimate the percentage of lymphocyte subpopulations in CSF. We hypothesized that an increased ratio of B-lineage cells in CSF of MS patients, as assessed with FC, could be useful for diagnostics. We analyzed CSF of 137 patients (70 MS, 24 infectious/autoimmune neurologic disorders (INDs), and 43 non-infectious/autoimmune neurologic disorders (NINDs)), and showed that CSF plasmablasts of >0.1% had a sensitivity of 40% for MS and specificity of 92% when comparing MS and IND, while plasmablasts of >0.25% had sensitivity of 36%, and 100% specificity.
Influence of Aging Processes on the Biology and Outcome of Multiple Myeloma [Meeting Abstract]
Introduction While Multiple myeloma (MM) is a disease of the elderly diagnosed at a median age of 69 years with nearly a third being above the age 75, little is known about the impact of aging processes on either disease biology or clinical outcomes. Treatment decisions are complicated, and it is important to take account three interacting variables: tumor genetics, comorbidities and the efficacy and toxicity of the treatment selected. While frailty scores help stratify elderly MM patients by functional status, quantitative measures of aging could provide biological markers to enhance clinical staging systems, standardize decision making, and guide treatment choices in the elderly MM population. In this work, we characterized the genetics of older MM patients compared to younger patients, and determined the associations of age with clonal hematopoiesis and telomere length (TL), both of which have been shown to be impacted by aging. Methods Using the MMRF CoMMpass IA15 data, we analyzed 972 NDMM patients with whole genome long insert sequencing with matching whole exomes. Using paired samples, we determined mutations (Mutect2 and Strelka), copy number (Control-FREEC v. 11.4), translocations (Manta v. 1.4.0), complex rearrangements (ChainFinder and ShatterSeek), as previously described and TL using Telomerecat. Looking at the germline data, we quantified Clonal Hematopoiesis of Indeterminate Potential (CHIP) and quantified TL using the same approach. Results The overall survival of patients aged over sixty-five is significantly worse than patients younger than this age (HR 1.7 (CI 95% 1.3-2.3), p<0.0001). Using a Bayesian approach, we show that, that del(16p) and del(6q) were more frequent in older patients (Corr=0.10, BF=1.1 and Corr=0.13, BF=11). Similarly, mutational signatures did not substantially differ between age groups with the exception of the proportion of APOBEC (SBS2 and 5) which was higher in the group over age > 80 (chi2=11, p=0.02). We determined both simple and complex structural variants and found that the prevalence of chromothripsis increased with age (chi2=10.8, p=0.001). To determine whether this may be related to chromosomal instability occurring as a consequence of aging we examined the extent of telomere attrition. A significant negative correlation was identified between TL and age (F=9.5, p=0.002) but there were no correlations with complex rearrangements. We did, however, find that TL was significantly shorter in the TP53 (chi2=9, p=0.002) and ATM (chi2=7.2, p=0.007) mutated groups suggesting TL shortening may be associated with DNA instability. To further determine the association of short TL in malignant plasmacells with adverse outcomes we ranked patients based on TL quartile and determined the impact on outcome for the shortest TL. We show that 14%, 29%, 24%, 29%, and 21% of the >50, 50-60, 60-70-70-80, and >80 year old patients were within this short TL group. There was a significant correlation with adverse overall-survival both in the younger and older patients, Figure 1A. To understand and quantify the impact of aging of the normal hematopoietic system on outcomes in MM we quantified CHIP and TL on the germline samples. CHIP was seen in 156 patients (16%) and DNMT3A, ASXL1, and TET2 were the more frequent mutations. Patients with CHIP were significantly older (chi2=3.9, p=0.005), as it was seen in 22% of the over 80. The only signatures identified using a fitting approach for these CHIP mutations were the two age related mutational signatures (SBS1 and SBS5). Interestingly, patients with CHIP did not have significantly adverse clinical outcome. To understand the impact of genetics and markers of aging in the older population we performed a multivariate analysis on the subset of patients over age 65 (n=375). Like others, we found that the well described prognostic genetic risk factors (del(17p), TP53 mutations, t(4;14), t(14;16), and amp1q) did not appear to contribute to the independent assessment of risk when taking into account age, ISS, and performance status (ECOG>=2). We show that in this population of older myeloma that short TL was, however, an independent marker for negative outcome, Figure 1B.
Conclusion(s): We highlight the importance of TL, a composite factor that takes into account both DNA instability, copy number losses, and aging as a potential novel biological marker to assess outcome and aid personalized treatment decisions in older patients with MM. [Formula presented] Disclosures: Bauer: Synthekine: Current Employment. Braunstein: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Epizyme: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees. Landgren: Amgen: Consultancy, Honoraria, Research Funding; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Seattle Genetics: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Glenmark: Consultancy, Honoraria, Research Funding; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Binding Site: Consultancy, Honoraria; Cellectis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Karyopharma: Research Funding; BMS: Consultancy, Honoraria; Binding Site: Consultancy, Honoraria; Karyopharma: Research Funding; Merck: Other; Glenmark: Consultancy, Honoraria, Research Funding; Adaptive: Consultancy, Honoraria; Seattle Genetics: Research Funding; Pfizer: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Cellectis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Merck: Other. Davies: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotech: Honoraria; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Morgan: Karyopharm: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding.
A Case of CD5 Positive Hairy Cell Leukemia withBRAF V600EMutation and Lymph Node Involvement; Unique Case Report and Review of the Literature: [Meeting Abstract]
Objective: Hairy cell leukemia (HCL) is distinct indolent neoplasm of small mature B cells accounting for 2% of the lymphoid leukemias. The classic immunophenotypic profile of HCL includes bright expression of B cell surface antigen and immunoglobulins and lack of both CD5 and CD10. The tumor cells are predominantly present in the spleen, peripheral blood and bone marrow. Aberrant expression of CD5 and lymph node involvement are very rare. A case of CD5(+) HCL with a documentedBRAF V600Emutation has not been previously reported. This case is also unique due to lymph node involvement by HCL. To our knowledge, this is the only case report of a documentedBRAF V600Emutated CD5(+) HCL along with lymph node involvement in the literature.
Method(s): We are presenting a 45 years old man with multiple prior medical conditions including diabetes, COPD and interstitial pneomonitis with new onset of B-symptoms and neutropenia/monocytopenia. Peripheral blood flow cytometry revealed 21% CD5(+) CD10(-) monoclonal B cell population. Further workup revealed the neoplastic cells are positive for CD11c, CD103 and CD25. Bone marrow study revealed an extensive involvement of the marrow.By immunohistochemistry, the cells are also positive for CD5, CD20, BRAF and cyclin D1. Cytogenetic studies show that the cells are negative t(11;14) and molecular studies revealedBRAF V600Emutation. Reexamination of the prior wedge lung resection revealed intrathoracic lymph node involvement.
Result(s) and Conclusion(s): The most common differential diagnoses of a CD5(+) B cell lymphoma involving the bone marrow is chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. The key into the diagnosis of this case was marked monocytopenia that lead to addition of HCL antibody tubes despite the CD5 positivity and detection of CD25, CD103 and CD11c antigens in the leukemic cells. Bone marrow biopsy show extensive involvement by a CD5(+) cyclin D1(+) B cell lymphoma. The most important differential diagnosis in this case was mantle cell lymphoma. The diagnosis of HCL was confirmed by detection ofBRAF V600Emutation and absence of t (11;14). Retrospective examination of the intrathoracic lymph nodes show one lymph node involvement by hairy cell leukemia cells. The patient was treated with Vemurafenib and his follow up flow cytometry revealed only 0.60% leukemic involvement. Incorporation of clinical data at the time of flow cytometric examination is essential to aid the hematopathologist in having a broader differential diagnoses and addition of appropriate tests that lead to the more accurate diagnosis and treatment. [Formula presented] Disclosures: No relevant conflicts of interest to declare.
Epigenetic silencing of the ubiquitin ligase subunit FBXL7 impairs c-SRC degradation and promotes epithelial-to-mesenchymal transition and metastasis
Epigenetic plasticity is a pivotal factor that drives metastasis. Here, we show that the promoter of the gene that encodes the ubiquitin ligase subunit FBXL7 is hypermethylated in advanced prostate and pancreatic cancers, correlating with decreased FBXL7 mRNA and protein levels. Low FBXL7 mRNA levels are predictive of poor survival in patients with pancreatic and prostatic cancers. FBXL7 mediates the ubiquitylation and proteasomal degradation of active c-SRC after its phosphorylation at Serâ€‰104. The DNA-demethylating agent decitabine recovers FBXL7 expression and limits epithelial-to-mesenchymal transition and cell invasion in a c-SRC-dependent manner. In vivo, FBXL7-depleted cancer cells form tumours with a high metastatic burden. Silencing of c-SRC or treatment with the c-SRC inhibitor dasatinib together with FBXL7 depletion prevents metastases. Furthermore, decitabine reduces metastases derived from prostate and pancreatic cancer cells in a FBXL7-dependent manner. Collectively, this research implicates FBXL7 as a metastasis-suppressor gene and suggests therapeutic strategies to counteract metastatic dissemination of pancreatic and prostatic cancer cells.
Evaluation of novel t-follicular helper cell associated genes as biomarkers in t-cell neoplasms [Meeting Abstract]
Background: The 2017 WHO update on hematological malignancies recognizes lymphomas of follicular helper T-cell (TFH) origin which include angioimmunoblastic T-cell lymphoma (AITL) as well as the provisional entities follicular peripheral T-cell lymphoma (F-PTCL) and nodal PTCL with TFH phenotype (PTCL-TFH). Demonstration of TFH derivation requires expression of at least 2 or 3 antigens among CD279/PD1, CD10, BCL6, CXCL13, ICOS, SAP, and CCR5, but these markers are frequently not co-expressed. We hypothesized that evaluation of normal TFH cells may identify novel markers that may aid in identifying TFH-derived neoplasms as well as resolve potential biological heterogeneity amongst them.
Design(s): In order to identify potential novel TFH markers, we evaluated RNA-sequencing data generated from human TFH cells, T-effector (Teff), and naive T-cells available from the GEO database (GSE58596). Among differentially expressed genes (DEGs), a subset was assessed for their ability to stain a tissue microarray representing a variety of T-cell neoplasms using commercially available antibodies (TIM1, HES6, HDAC9, TGFBR3). Each marker was scored by at least two pathologists for staining intensity and frequency of neoplastic Tcells in two separate cores to obtain an H-score; an H-score > 5 was defined as positive.
Result(s): We identified 132 genes (p value <0.05) highly expressed in TFH cells compared to both Teff and naive T-cells; these genes did not include CD279/PD1, CD10, BCL6, CXCL13, ICOS, SAP, or CCR5. While HES6 and TIM1 immunostains highlighted cells similar in number and distribution to established TFH markers such as PD1, ICOS, and CXCL13 in normal lymphoid tissue, HDAC9 and TGFBR3 were more broadly expressed. TGFBR3 was frequently expressed in AITL, MF, and ALK- ALCL, but was expressed in a smaller frequency of PTCL, NOS cases. While TIM1 and TGFBR3 highlighted the majority of AITL and PTCL, NOS cases, small subsets of AITL and PTCL, NOS cases expressed only TIM1 or TGFBR3. A summary of staining frequencies for each candidate TFH marker is shown in the table below. (Figure presented)
Conclusion(s): Overall, these findings suggest that TGFBR3 and TIM1 may be used as TFH markers and also suggest that presumed TFHderived neoplasms exhibit additional biological heterogeneity with respect to TFH marker expression. A more detailed comparison of TIM1 and TGFBR3 to commonly used TFH markers will be required to confirm the utility of these markers in the clinical diagnostic setting
A simple two-step test based on csf flow cytometry helps to discriminate ms from other inflammatory and noninflammatory neurologic disorders [Meeting Abstract]