Transcriptional control of CBX5 by the RNA binding proteins RBMX and RBMXL1 maintains chromatin state in myeloid leukemia.

RNA binding proteins (RBPs) are key arbiters of post-transcriptional regulation and are found to be found dysregulated in hematological malignancies. Here, we identify the RBP RBMX and its retrogene RBMXL1 to be required for murine and human myeloid leukemogenesis. RBMX/L1 are overexpressed in acute myeloid leukemia (AML) primary patients compared to healthy individuals, and RBMX/L1 loss delayed leukemia development. RBMX/L1 loss lead to significant changes in chromatin accessibility, as well as chromosomal breaks and gaps. We found that RBMX/L1 directly bind to mRNAs, affect transcription of multiple loci, including CBX5 (HP1α), and control the nascent transcription of the CBX5 locus. Forced CBX5 expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through their downstream target CBX5. These findings identify a mechanism for RBPs directly promoting transcription and suggest RBMX/L1, as well as CBX5, as potential therapeutic targets in myeloid malignancies.

RNA Regulators in Leukemia and Lymphoma.

Posttranscriptional regulation of mRNA is a powerful and tightly controlled process in which cells command the integrity, diversity, and abundance of their protein products. RNA-binding proteins (RBPs) are the principal players that control many intermediary steps of posttranscriptional regulation. Recent advances in this field have discovered the importance of RBPs in hematological diseases. Herein we will review a number of RBPs that have been determined to play critical functions in leukemia and lymphoma. Furthermore, we will discuss the potential therapeutic strategies that are currently being studied to specifically target RBPs in these diseases.

HyperTRIBE uncovers increased MUSASHI-2 RNA binding activity and differential regulation in leukemic stem cells.

The cell-context dependency for RNA binding proteins (RBPs) mediated control of stem cell fate remains to be defined. Here we adapt the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to globally map the mRNA targets of the RBP MSI2 in mammalian adult normal and malignant stem cells. We reveal a unique MUSASHI-2 (MSI2) mRNA binding network in hematopoietic stem cells that changes during transition to multipotent progenitors. Additionally, we discover a significant increase in RNA binding activity of MSI2 in leukemic stem cells compared with normal hematopoietic stem and progenitor cells, resulting in selective regulation of MSI2's oncogenic targets. This provides a basis for MSI2 increased dependency in leukemia cells compared to normal cells. Moreover, our study provides a way to measure RBP function in rare cells and suggests that RBPs can achieve differential binding activity during cell state transition independent of gene expression.

Functional screen of MSI2 interactors identifies an essential role for SYNCRIP in myeloid leukemia stem cells.

The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia.

MSI2 is required for maintaining activated myelodysplastic syndrome stem cells.

Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease.

Avaliação do índice proliferativo dos carcinomas da mama: aspectos técnicos e utilidade clínica / Evaluation of proliferative index of breast carcinoma: technical aspects and clinical utility

INTRODUÇÃO: A avaliação do índice proliferativo pela expressão do Ki67 por imuno-histoquímica (IP-Ki67) é uma importante ferramenta prognóstica em alguns tumores. No câncer de mama seu uso ainda é opcional devido a discussões sobre o seu valor prognóstico, valor preditivo e o melhor ponto de corte. MÉTODOS: Estudo retrospectivo para avaliar os métodos de quantificação do IP-KI67 (estimativa global, contagem diferencial de células e analise digital automatizada), diferentes pontos de corte, o valor preditivo e prognóstico no carcinoma invasor da mama (CDI). RESULTADOS: O IP-KI67 avaliado pelos diferentes métodos mostrou ótima correlação direta variando entre 0,86 e 0,95 (p<0,0001). O índice Kappa entre as metodologias variou de moderada a excelente para o ponto de corte de 14% variou entre 0,53 e 0,87 (p<0,0001) e o índice Kappa ponderado para os grupos <10%, 10-20% e >20% variou entre 0,46 e 0,85 (p<0,0001). A sobrevida livre de doença em 10 anos foi de 54,9%, 43,7%, 43,8% para o IP-KI67 <10%, 10%-20% e >20%, respectivamente (p=0,0373). A melhor sobrevida em 10 anos foi observada mesmo nos pacientes com doença em estágio clínico I e IIA (87,5% vs 80,2% vs 30,2%, respectivamente, p=0,0001). Pacientes com CDI em estágio precoce (axila negativa) com ou sem quimioterapia e IP-KI67 muito baixo, exibiram curvas de sobrevida livre de doença similares em 10 anos (p=0,3842). A variável IP-KI67 não foi capaz de identificar as pacientes com tumores luminais sensiveis a quimioterapia neoadjuvante. CONCLUSÃO: O IP-KI67 avaliado pela estimativa global do patologista tem boa correlação com a quantificação digital automatizada. A biopsia por agulha grossa é um espécime adequado para avaliar o IP-KI67. O IP-KI67 não tem valor prognóstico para sobrevida livre de doença em 5 e 10 anos. O valor preditivo de resposta a quimioterapia neoadjuvante do IP-KI67 é limitado com uso dos pontos de corte atualmente sugeridos pela literatura e não deve ser considerado para a indicação do tratamento.

INTRODUCTION: Evaluation of tumor proliferation by Ki67 expression using immunohistochemistry (IP-Ki67) has been studied as a prognostic and predictive tool in some tumors. In breast cancer, its use remains optional due to discussions on best cutoff and methods of quantification. METHODS: We performed a retrospective study to evaluate distinct methods of quantification of Ki67-IP (global estimate differential cell count and automated digital processing), different cutoff points, the predictive and prognostic value in invasive breast carcinoma (IBC). RESULTS: The IP-Ki67 assessed by different methods showed excellent correlation, (r between 0.86 and 0.95 (p <0.0001). Kappa Index between methodologies for the cut-off of 14% ranged from 0.53 to 0.87 (p <0.0001) and the weighted Kappa index for groups <10%, 10-20% and> 20% ranged between 0.46 and 0.85 (p <0.0001). Disease-free survival (DFS) at 10 years was 54,9%,43,7%, 43,8% for IP-KI67 <10%, 10%-20% e >20% , respectively (p = 0.0373) but it was highly associated with traditional prognostic factors. IBC patients in early stage (negative axilla) with or without chemotherapy and very low IP-Ki67 showed similar 10y-DFS compared to higher IP-KI67 (p = 0.3842). IP-Ki67 did not identify a subgroup of patients with luminal tumors with higher sensitivity to neoadjuvant chemotherapy. CONCLUSION: The IP- Ki67 assessed by overall has good correlation to automated digital quantification. Core needle biopsy is adequate for IP-Ki67 evaluation. The IP-KI67 is not independent prognostic factor for IBC. The predictive value is very limited and should not be used to indicate the neoadjuvant treatment