Ramakrishna Sompallae

18 F-FDG-PET/CT imaging in an IL-6- and MYC-driven mouse model of human multiple myeloma affords objective evaluation of plasma cell tumor progression and therapeutic response to the proteasome inhibitor ixazomib

18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT) are useful imaging modalities for evaluating tumor progression and treatment responses in genetically engineered mouse models of solid human cancers, but the potential of integrated FDG-PET/CT for assessing tumor development and new interventions in transgenic mouse models of human blood cancers such as multiple myeloma (MM) has not been demonstrated. Here we use BALB/c mice that contain the newly developed iMycΔEμ gene insertion and the widely expressed H2-Ld-IL6 transgene to demonstrate that FDG-PET/CT affords an excellent research tool for assessing interleukin-6- and MYC-driven plasma cell tumor (PCT) development in a serial, reproducible and stage- and lesion-specific manner. We also show that FDG-PET/CT permits determination of objective drug responses in PCT-bearing mice treated with the investigational proteasome inhibitor ixazomib (MLN2238), the biologically active form of ixazomib citrate (MLN9708), that is currently in phase 3 clinical trials in MM. Overall survival of 5 of 6 ixazomib-treated mice doubled compared with mice left untreated. One outlier mouse presented with primary refractory disease. Our findings demonstrate the utility of FDG-PET/CT for preclinical MM research and suggest that this method will play an important role in the design and testing of new approaches to treat myeloma.

Phenotypic and Functional Alterations in Circulating Memory CD8 T Cells with Time after Primary Infection

Memory CD8 T cells confer increased protection to immune hosts upon secondary viral, bacterial, and parasitic infections. The level of protection provided depends on the numbers, quality (functional ability), and location of memory CD8 T cells present at the time of infection. While primary memory CD8 T cells can be maintained for the life of the host, the full extent of phenotypic and functional changes that occur over time after initial antigen encounter remains poorly characterized. Here we show that critical properties of circulating primary memory CD8 T cells, including location, phenotype, cytokine production, maintenance, secondary proliferation, secondary memory generation potential, and mitochondrial function change with time after infection. Interestingly, phenotypic and functional alterations in the memory population are not due solely to shifts in the ratio of effector (CD62Llo) and central memory (CD62Lhi) cells, but also occur within defined CD62Lhi memory CD8 T cell subsets. CD62Lhi memory cells retain the ability to efficiently produce cytokines with time after infection. However, while it is was not formally tested whether changes in CD62Lhi memory CD8 T cells over time occur in a cell intrinsic manner or are due to selective death and/or survival, the gene expression profiles of CD62Lhi memory CD8 T cells change, phenotypic heterogeneity decreases, and mitochondrial function and proliferative capacity in either a lymphopenic environment or in response to antigen re-encounter increase with time. Importantly, and in accordance with their enhanced proliferative and metabolic capabilities, protection provided against chronic LCMV clone-13 infection increases over time for both circulating memory CD8 T cell populations and for CD62Lhi memory cells. Taken together, the data in this study reveal that memory CD8 T cells continue to change with time after infection and suggest that the outcome of vaccination strategies designed to elicit protective memory CD8 T cells using single or prime-boost immunizations depends upon the timing between antigen encounters.

The tumor microenvironment is the main source of IL-6 for plasma cell tumor development in mice

The tumor microenvironment is the main source of IL-6 for plasma cell tumor development in mice

FOXM1 is a therapeutic target for high-risk multiple myeloma

The transcription factor forkhead box M1 (FOXM1) is a validated oncoprotein in solid cancers, but its role in malignant plasma cell tumors such as multiple myeloma (MM) is unknown. We analyzed publicly available MM data sets and found that overexpression of FOXM1 prognosticates inferior outcome in a subset (~15%) of newly diagnosed cases, particularly patients with high-risk disease based on global gene expression changes. Follow-up studies using human myeloma cell lines (HMCLs) as the principal experimental model system demonstrated that enforced expression of FOXM1 increased growth, survival and clonogenicity of myeloma cells, whereas knockdown of FOXM1 abolished these features. In agreement with that, constitutive upregulation of FOXM1 promoted HMCL xenografts in laboratory mice, whereas inducible knockdown of FOXM1 led to growth inhibition. Expression of cyclin-dependent kinase 6 (CDK6) and NIMA-related kinase 2 (NEK2) was coregulated with FOXM1 in both HMCLs and myeloma patient samples, suggesting interaction of these three genes in a genetic network that may lend itself to targeting with small-drug inhibitors for new approaches to myeloma therapy and prevention. These results establish FOXM1 as high-risk myeloma gene and provide support for the design and testing of FOXM1-targeted therapies specifically for the FOXM1High subset of myeloma.

Anchored multiplex PCR for targeted next-generation sequencing reveals recurrent and novel USP6 fusions and upregulation of USP6 expression in aneurysmal bone cyst

Primary aneurysmal bone cyst (ABC) is a neoplastic process due to recurrent translocations involving the USP6 gene. By fluorescence in situ hybridization, up to 69% of primary ABCs harbored USP6 translocations; no USP6 translocation was found in secondary ABC or giant cell tumor of bone (GCT). GCT can recur locally, metastasize to the lungs in some cases, and rarely undergo malignant transformation. Differentiating primary ABC from its mimics is important for treatment and prognosis. We evaluated USP6 fusion and expression in 13 cases of primary and 1 case of secondary ABC, and 9 cases of GCT using nucleic acid extracted from formalin‐fixed, paraffin‐embedded tissue and a next generation sequencing (NGS)‐based assay. USP6 fusions including 7 novel fusions and USP6 transcripts were identified in all 13 primary ABCs. Nine cases with strong evidence of fusions showed high levels of USP6 transcripts by reverse transcription‐PCR (RT‐PCR). The remaining four had no detectable USP6 expression by a first‐round of RT‐PCR but the presence of USP6 transcripts was identified by a second‐round, nested PCR. The major fusions were confirmed by RT‐PCR followed by Sanger sequencing. No USP6 fusion or transcript was detected in any of the GCTs or the case of secondary ABC by NGS or by two rounds of PCR. All USP6 translocations resulted in fusion of the entire USP6 coding sequence with promoters of the fusion gene leading to upregulation of USP6 transcription, which is likely the underlying mechanism for ABC oncogenesis.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-γ and IL-17 production in blood from TB + patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras

BackgroundA better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates.MethodsRecombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-γ and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29).ResultsM.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-γ production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c.ConclusionsThe pattern of immune target recognition is different in regard to IFN-γ and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.

TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF

Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the “master regulator” of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.

The NAB2–STAT6 gene fusion in solitary fibrous tumor can be reliably detected by anchored multiplexed PCR for targeted next-generation sequencing

Solitary fibrous tumor (SFT) is a mesenchymal tumor of fibroblastic origin, which can affect any region of the body. 10-15% of SFTs metastasize and metastatic tumors are uniformly lethal with no effective therapies. The behavior of SFT is difficult to predict based on morphology. Recently, an intrachromosomal gene fusion between NAB2 and STAT6 was identified as the defining driving genetic event of SFT and different fusion types correlated with tumor histology and behavior. Due to the proximity of NAB2 and STAT6 on chromosome 12, this fusion may be missed by fluorescence in-situ hybridization. We evaluated 12 SFTs from 10 patients. All tumors showed strong nuclear staining for STAT6 by immunohistochemistry (IHC). The same formalin-fixed, paraffin-embedded blocks for IHC were used for gene fusion detection by a next-generation sequencing (NGS)-based assay. Targeted RNA fusion sequencing for gene fusions was performed using the Universal RNA Fusion Detection Kit, the Archer(™) FusionPlex(™) Sarcoma Panel and the Ion Torrent PGM, and data were analyzed using the Archer Analysis Pipeline 3.3. All tumors were positive for NAB2-STAT6 fusion. Six types of fusions were detected: NAB2ex4-STAT6ex2, NAB2ex2-STAT6ex5, NAB2ex6-STAT6ex16, NAB2ex6-STAT6ex17, NAB2ex3-STAT6ex18 and NAB2intron6-STAT6Ex17. The NGS findings were confirmed by RT-PCR followed by Sanger sequencing. No STAT6 fusion was detected in selected morphologic mimics of SFT. The assay also allows for detection of novel fusions and can detect NAB2-STAT6 fusions at a single-base resolution.

Upregulation of FOXM1 in a subset of relapsed myeloma results in poor outcome

Following up on our previous work demonstrating the involvement of the transcription factor, forkhead box M1 (FOXM1), in the biology and outcome of a high-risk subset of newly diagnosed multiple myeloma (nMM), we sought to determine whether upregulation of FOXM1 may also be a feature of relapsed myeloma (rMM). To that end, we analyzed the total therapy 2 (TT2) dataset (GSE2658) from the University of Arkansas for Medical Science, which contains microarray-based gene expression and clinical outcome data on 88 patients with known FOXM1 mRNA levels at baseline and relapse. Statistical comparison demonstrated a significant increase (p= 10) in mean (by ~70%) and median (~2.9-fold) FOXM1 expression upon relapse (Fig. 1a, left). Just like in nMM, the distinction between high and low FOXM1 message at relapse was of prognostic value: FOXM1 patients (n= 11, 12.5%) exhibited markedly reduced event-free survival and overall survival compared to FOXM1 patients (n = 77, Fig. 1a, right). To confirm these results with the help of an independent dataset that is more recent than TT2, we took advantage of myeloma patients from the University of Heidelberg, Germany, which were uniformly treated upfront using HDT/ASCT and new myeloma drugs. In this cohort of 692 patients with nMM, FOXM1 status was as negative a predictor of survival (Fig. 1b) as in the original study. Furthermore, relapsed myelomas from the Heidelberg cohort (n= 55) also demonstrated significant elevations of FOXM1 message compared to nMM (Fig. 1c, left) and FOXM1 status at relapse—found in 8 of 60 (13%) patients—led to an even more dramatic reduction in survival than seen in the TT2 cohort (Fig. 1c, right). These findings led us to conclude that upregulation of FOXM1 is a molecular marker of a subset of relapsed myelomas that results in inferior outcome in patients with peak expression levels. Because (1) FOXM1 is best known as positive regulator of cell cycle progression, (2) tumor cell proliferation is an important, independent, adverse determinant of myeloma patient survival and (3) myeloma relapse from chemotherapy ultimately results in selection of cell clones with increased proliferation rates, we decided to evaluate whether increased FOXM1 expression at relapse may be associated with increased myeloma proliferation. For this purpose, we relied on a validated gene expression-based proliferation index (GPI) of myeloma to stratify 712 patients with nMM and 82 patients with rMM from the Heidelberg cohort into three different groups defined by low, intermediate or high proliferation rates of tumor cells. Fig. 1d shows that FOXM1 expression paralleled the GPI score in both nMM (left panel) and rMM (right panel). High FOXM1 message levels, which even surpassed those measured in GPI myeloma, were also observed in normal plasmablasts derived from cytokineinduced peripheral blood B-lymphocytes of healthy donors and in rapidly dividing HMCLs (human myeloma cell lines) propagated in vitro (Fig. 1e). These results were consistent with the contention that FOXM1 regulates cell cycle progression in both normal and neoplastic plasma cells. Recurrent cancer including relapsed myeloma is frequently associated with therapy resistance acquired by changes in genetic pathways wherein FOXM1 has been repeatedly implicated. With this in mind, we asked

Virus-induced inflammasome activation is suppressed by prostaglandin D2/DP1 signaling

Significance Inflammatory responses to viral infections must be optimized to clear the pathogen without tissue damage. Inflammasomes comprise an important component of the innate immune response. Inflammasome activity must be carefully controlled to prevent a hyperinflammatory response, especially in brain infections. Here we identify a host factor, PYDC3, that is dependent upon prostaglandin D2 (PGD2) and IFN-I signaling and is required to modulate inflammasome activation. After infection, inflammasome activation and expression of a downstream proinflammatory cytokine, IL-1β, were increased in mice deficient in PGD2 signaling, decreasing survival. Excess mortality was reversed by IL-1β receptor blockade. These results define a consequence of prostaglandin signaling and shed light on prostaglandin–inflammasome interactions, which modulate excessive inflammation and tissue damage in the virus-infected brain. Prostaglandin D2 (PGD2), an eicosanoid with both pro- and anti-inflammatory properties, is the most abundantly expressed prostaglandin in the brain. Here we show that PGD2 signaling through the D-prostanoid receptor 1 (DP1) receptor is necessary for optimal microglia/macrophage activation and IFN expression after infection with a neurotropic coronavirus. Genome-wide expression analyses indicated that PGD2/DP1 signaling is required for up-regulation of a putative inflammasome inhibitor, PYDC3, in CD11b+ cells in the CNS of infected mice. Our results also demonstrated that, in addition to PGD2/DP1 signaling, type 1 IFN (IFN-I) signaling is required for PYDC3 expression. In the absence of Pydc3 up-regulation, IL-1β expression and, subsequently, mortality were increased in infected DP1−/− mice. Notably, survival was enhanced by IL1 receptor blockade, indicating that the effects of the absence of DP1 signaling on clinical outcomes were mediated, at least in part, by inflammasomes. Using bone marrow-derived macrophages in vitro, we confirmed that PYDC3 expression is dependent upon DP1 signaling and that IFN priming is critical for PYDC3 up-regulation. In addition, Pydc3 silencing or overexpression augmented or diminished IL-1β secretion, respectively. Furthermore, DP1 signaling in human macrophages also resulted in the up-regulation of a putative functional analog, POP3, suggesting that PGD2 similarly modulates inflammasomes in human cells. These findings demonstrate a previously undescribed role for prostaglandin signaling in preventing excessive inflammasome activation and, together with previously published results, suggest that eicosanoids and inflammasomes are reciprocally regulated.