Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy.

RhoH is an unusual member of the Rho family of small GTP-binding proteins in that it lacks GTPase activity. Since the RhoH protein is constantly bound by GTP, it is constitutively active and controlled predominantly by changes in quantitative expression. Abnormal levels of RHOH gene transcripts have been linked to a range of malignancies including acute myeloid leukemia (AML). One of the hallmarks of AML is a block in the normal program of myeloid differentiation. Here we investigate how myeloid differentiation is controlled by the quantitative expression of RHOH. Our analysis demonstrates that increasingly mature myeloid cells express progressively lower levels of RHOH. However, as monocytic myeloid cells terminally differentiate into macrophages, RHOH expression is up-regulated. This up-regulation is not apparent in AML where myeloid differentiation is blocked at stages of low RHOH expression. Nevertheless, when the up-regulation of RHOH is forced, then terminal macrophage differentiation is induced and the Cdc42 and Wnt intracellular signalling pathways are repressed. These results indicate that RHOH induction is a driver of terminal differentiation and might represent a means of effecting AML differentiation therapy. The potential of this therapeutic strategy is supported by forced up-regulation of RHOH reducing the ability of AML cells to produce tumours in vivo.

Aggressiveness Potential of Spontaneous Canine Mucosal Melanoma Can Dictate Distinct Cancer Stem Cell Compartment Behaviors in Regard to Their Initial Size and Expansion Abilities.

Mucosal melanoma represents one of the most highly metastatic and aggressive subtypes of melanoma. The biology of mucosal melanoma is poorly documented, and the lack of experimental models makes it difficult to design and test new therapies. Dogs are frequently affected by melanomas of the oral cavity, making spontaneous canine melanoma a potentially predictable model for their human counterpart. We recently established and characterized two new canine mucosal melanoma cell lines named OCR_OCMM1 and OCR_OCMM2. Here, we identified quiescent cancer stem cell (CSC) subpopulations in both canine cell lines that displayed similarities to human quiescent CSCs: canine melanoma CSCs had the ability to self-renew, produced nonstem cell (SC) progeny, and formed melanospheres that recapitulated the phenotypic profile of the parental tumor. These CSCs also formed melanoma in immunodeficient mice, and the inhibition of PI3K/AKT signaling expanded the CSC pool. A subset of non-CSCs transitioned to become CSCs. OCR_OCMM1 and OCR_OCMM2 displayed different CSC compartment behaviors in regard to their initial size and expansion abilities. Collectively, this study showed that the OCR_OCMM1 and OCR_OCMM2 canine melanoma cell lines are powerful cellular tools to study melanoma SCs, not only for mucosal but also for the more common human cutaneous melanoma.

Isolation and characterization of two canine melanoma cell lines: new models for comparative oncology.

BACKGROUND: Metastatic melanoma is one of the most aggressive forms of cancer in humans. Among its types, mucosal melanomas represent one of the most highly metastatic and aggressive forms, with a very poor prognosis. Because they are rare in Caucasian individuals, unlike cutaneous melanomas, there has been fewer epidemiological, clinical and genetic evaluation of mucosal melanomas. Moreover, the lack of predictive models fully reproducing the pathogenesis and molecular alterations of mucosal melanoma makes its treatment challenging. Interestingly, dogs are frequently affected by melanomas of the oral cavity that are characterized, as their human counterparts, by focal infiltration, recurrence, and metastasis to regional lymph nodes, lungs and other organs. In dogs, some particular breeds are at high risk, suggesting a specific genetic background and strong genetic drivers. Altogether, the striking homologies in clinical presentation, histopathological features, and overall biology between human and canine mucosal melanomas make dogs invaluable natural models with which to investigate tumor development, including tumor ætiology, and develop tailored treatments. METHODS: We developed and characterized two canine oral melanoma cell lines from tumors isolated from dog patients with distinct clinical profiles; with and without lung metastases. The cells were characterized using immunohistochemistry, pharmacology and genetic studies. RESULTS: We have developed and immunohistochemically, genetically, and pharmacologically characterized. Two cell lines (Ocr_OCMM1X & Ocr_OCMM2X) were produced through mouse xenografts originating from two clinically contrasting melanomas of the oral cavity. Their exhaustive characterization showed two distinct biological and genetic profiles that are potentially linked to the stage of malignancy at the time of diagnosis and sample collection of each melanoma case. These cell lines thus constitute relevant tools with which to perform genetic and drug screening analyses for a better understanding of mucosal melanomas in dogs and humans. CONCLUSIONS: The aim of this study was to establish and characterize xenograft-derived canine melanoma cell lines with different morphologies, genetic features and pharmacological sensitivities that constitute good predictive models for comparative oncology. These cell lines are relevant tools to advance the use of canine mucosal melanomas as natural models for the benefit of both veterinary and human medicine.

BACH2 promotes indolent clinical presentation in Waldenström macroglobulinemia.

Approximately 30% of the patients who fulfil the criteria of Waldenström's macroglobulinemia (WM) are diagnosed while asymptomatic (indolent), and will not require immediate therapy. Conversely, patients with a disease-related event will be considered for therapy. The physiopathology of these 2 groups remains unclear, and the mechanisms of progression from indolent to symptomatic WM have yet to be fully understood. Seventeen patients diagnosed with WM were included in this study, 8 asymptomatic WM (A-WM) and 9 symptomatic WM (S-WM). A differential analysis was performed on a first series of 11 patients and identified 48 genes whose expression separated samples from A- to S-WM. This gene signature was then confirmed on a second independent validation set of 6 WM. Within this expression profile, BACH2, a B-cell transcription factor known to be a tumor suppressor gene, was found to be over-expressed in A-MW relatively to S-MW. We specifically over-expressed BACH2 in a WM-related cell line and observed a significant reduction of the clonogenic activity. To the best of our knowledge, we report for the first time a specific gene expression signature that differentiates A-WM and S-WM. Within this expression profile, BACH2 was identified as a candidate gene that may help to understand better the behavior of tumor cells in indolent WM.

CD38 in Hairy Cell Leukemia Is a Marker of Poor Prognosis and a New Target for Therapy.

Hairy cell leukemia (HCL) is characterized by underexpression of the intracellular signaling molecule RhoH. Reconstitution of RhoH expression limits HCL pathogenesis in a mouse model, indicating this could represent a new therapeutic strategy. However, while RhoH reconstitution is theoretically possible as a therapy, it is technically immensely challenging as an appropriately functional RhoH protein needs to be specifically targeted. Because of this problem, we sought to identify druggable proteins on the HCL surface that were dependent upon RhoH underexpression. One such protein was identified as CD38. Analysis of 51 HCL patients demonstrated that 18 were CD38-positive. Interrogation of the clinical record of 23 relapsed HCL patients demonstrated those that were CD38-positive had a mean time to salvage therapy 71 months shorter than patients who were CD38-negative. Knockout of the CD38 gene in HCL cells increased apoptosis, inhibited adherence to endothelial monolayers, and compromised ability to produce tumors in vivo. Furthermore, an anti-CD38 antibody proved effective against pre-existing HCL tumors. Taken together, our data indicate that CD38 expression in HCL drives poor prognosis by promoting survival and heterotypic adhesion. Our data also indicate that CD38-positive HCL patients might benefit from treatments based on CD38 targeting.

Genome wide SNP array identified multiple mechanisms of genetic changes in Waldenstrom macroglobulinemia.

SNP array (SNPa) was developed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) without copy number changes, CN-LOH. We aimed to identify novel genomic aberrations using SNPa in 31 WM with paired samples. Methylation status and mutation were analyzed on target genes. A total of 61 genetic aberrations were observed, 58 CNA (33 gains, 25 losses) in 58% of patients and CN-LOH in 6% of patients. The CNA were widely distributed throughout the genome, including 12 recurrent regions and identified new cryptic clonal chromosomal lesions that were mapped. Gene set expression analysis demonstrated a relationship between either deletion 6q or gain of chromosome 4 and alteration of gene expression profiling. We then studied methylation status and sought for mutations in altered regions on target genes. We observed methylation of DLEU7 on chromosome 13 in all patients (n = 12) with WM, and mutations of CD79B/CD79A genes (17q region), a key component of the BCR pathway, in 15% of cases. Most importantly, higher frequency of ≥3 CNA was observed in symptomatic WM. In conclusion, this study expands the view of the genomic complexity of WM, especially in symptomatic WM, including a potentially new mechanism of gene dysfunction, acquired uniparental disomy/CN-LOH. Finally, we have identified new potential target genes in WM, such as DLEU7 and CD79A/B.

MYD88 L265P mutation in Waldenstrom macroglobulinemia.

Mutation of the MYD88 gene has recently been identified in activated B-cell-like diffuse cell lymphoma and enhanced Janus kinase/signal transducer and activator of transcription (JAK-STAT) and nuclear factor κB (NF-κB) signaling pathways. A whole exome-sequencing study of Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. The genetic background is not fully deciphered in WM, although the role of NF-κB and JAK-STAT has been demonstrated. We analyzed MYD88 mutation in exon 5 and characterized the clinical significance of this genetic alteration in 67 WM patients. Clinical features; immunophenotypic markers; and conventional cytogenetic, fluorescence in situ hybridization, and single nucleotide polymorphism array data were analyzed. MYD88 L265P mutation was acquired in 79% of patients. Overall, we have identified alteration of the MYD88 locus in 91% of WM patients, including 12% with gain on chromosome 3 at the 3p22 locus that included the MYD88 gene. Patients with absence of MYD88 mutation were WM characterized with a female predominance, a splenomegaly, gain of chromosome 3, and CD27 expression. Importantly, inhibition of MYD88 signaling induced cytotoxicity and inhibited cell growth of cell lines issued from patients with WM. In conclusion, these results confirm a high frequency of MYD88 L265P mutation in WM. The discovery of MYD88 L265P mutation may contribute to a better understanding of the physiopathogeny of WM.

Repression of the RHOH gene by JunD.

RhoH is a member of the Rho family of small GTP-binding proteins that lacks GTPase activity. Since RhoH is constantly bound by GTP, it is thought to be constitutively active and controlled predominantly by changes in quantitative expression. RhoH is produced specifically in haematopoietic cells and aberrant expression has been linked to various forms of leukaemia. Transcription of the RHOH gene is the first level at which the quantitative levels of the RhoH protein are regulated. Previous studies have demonstrated that RHOH gene transcription is initiated by three distinct promoter regions designated P1, P2 and P3 that define the 5' end of exons 1, 2 and 4 respectively. In the present study we report that the P3 promoter is largely responsible for RHOH gene transcription in the B-lymphocytic cell line Raji. The P3 promoter contains a minimal promoter region and a repressor region extending from -236 to +67 and +68 to +245 respectively, relative to the 5' end of exon 4. Chromatin immunoprecipitation demonstrated that two AP1 (activator protein 1) sites in the minimal promoter region bind JunD. When JUND is overexpressed, the endogenous RHOH gene is repressed; however, when JUND is inhibited, expression of endogenous RHOH is induced both in the Raji cell line and AML (acute myeloid leukaemia) cells. In the HCL (hairy cell leukaemia) cell line JOK-1, induction of RHOH increases expression of the α isoform of protein kinase C. This downstream target of RHOH is also induced in AML cells by JUND inhibition. Collectively, these data indicate that JunD is an inhibitor of RHOH gene expression.

High-throughput genomic analysis in Waldenström's macroglobulinemia.

Single-nucleotide polymorphism array (SNPa) and array-based comparative genomic hybridization (aCGH) are among the most sensitive genomic high-throughput screening techniques used in the exploration of genetic abnormalities in Waldenström's macroglobulinemia (WM). SNP and aCGH allow the identification of copy number abnormalities (CNA) at the kilobase level thus identifying cryptic genetic abnormalities unseen by lower-resolution approaches such as conventional cytogenetic or fluorescence in situ hybridization (FISH). CNA were identified in nearly 80% of cases by aCGH that delineated in addition minimal altered regions. At gene level, remarkable findings affecting genes involved in the regulation of the NF-kB signaling pathways were identified, such as biallelic inactivation of TNFAIP3 and TRAF3. SNPa also allowed characterization of copy neutral losses such as uniparental disomies (UPD), which is an important and frequent mechanism of gene alteration in cancer cells. Herein, we summarize the current knowledge of WM genomic basis using these high-throughput techniques.

Underexpression of RhoH in Hairy Cell Leukemia.

The cause of hairy cell leukemia (HCL) is unknown. Current treatments seem effective only for a limited period of time. In addition, a significant proportion of patients remain refractive to all treatment options. These considerations indicate the need to develop alternative therapeutic strategies for HCL. Here, we report that HCL is characterized by underexpression of RhoH. In vitro reconstitution of RhoH expression inhibits the aberrant adhesion and transendothelial migration that drives disease pathogenesis. In an in vivo model of HCL, RhoH reconstitution limits malignant progression and protects against mortality. These findings provide the proof of principle that RhoH reconstitution represents a potential new approach to the treatment of HCL.

Alteration of SMRT tumor suppressor function in transformed non-Hodgkin lymphomas.

Indolent non-Hodgkin lymphomas are characterized by a prolonged phase that is typically followed by a clinical progression associated with an accelerated clinical course and short survival time. Previous studies have not identified a consistent cytogenetic or molecular abnormality associated with transformation. The development of a transformed phenotype, evolving from the original low-grade component, most likely depends on multiple genetic events, including the activation of synergistic dominant oncogenes and a loss of tumor suppressor gene functions. Complex karyotypes and relatively bad chromosome morphology are typical of transformed non-Hodgkin lymphomas, rendering complete cytogenetic analysis difficult. Here, we report the use of transformed non-Hodgkin lymphoma cell lines and primary samples to identify the involvement of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) gene that maps at chromosome 12q24 in transformed non-Hodgkin lymphomas. We also show that down-regulation of SMRT in the immortalized "Weinberg's model" cell lines induces transformation of the cells. Assessment of cDNA array profiles should further help us to design a working model for SMRT involvement in non-Hodgkin lymphoma transformation as a novel, nonclassical tumor suppressor.

Structural features of hematopoiesis-specific RhoH/ARHH gene: high diversity of 5'-UTR in different hematopoietic lineages suggests a complex post-transcriptional regulation.

The hematopoiesis-specific RhoH gene is thought to be deregulated in B-cell non-Hodgkin's lymphoma (B-NHL), by either a chromosomal translocation or mutations, which affect its 5' regulatory region. The encoded Rho protein, always GTP-bound in vivo, was hypothesized to behave as a Rac antagonist. Extensive expression analysis allowed the detection of RhoH transcripts in all hematopoietic lineages (lymphoid, erythroid, myeloid), with a high level in lymphoid cells. To initiate investigations on the molecular mechanisms that regulate RhoH gene expression, Race-PCR and primer extension were conducted in the B-cell line Raji, which allowed (i) the establishment of RhoH complex intron/exon organization and (ii) the detection of several transcription initiation sites. In addition, a high 5' end heterogeneity of RhoH mRNAs was observed, due to alternative splicing of some 5' exons and to the use of these different transcription start sites. RT-PCR analysis led to the identification of this 5' end heterogeneity in different hematopoietic lineages. Discrepancies were particularly observed between B and T cells, due to an alternative splicing of one 5' exon (1b), which might be an important element in RhoH gene regulation. Such specific features have never been described for any Rho family member gene. They provide a molecular basis to study complex mechanisms involved in the control of RhoH expression.