Mouse models of chronic lymphocytic leukemia and Richter transformation: what we have learnt and what we are missing

Although the chronic lymphocytic leukemia (CLL) treatment landscape has changed dramatically, unmet clinical needs are emerging, as CLL in many patients does not respond, becomes resistant to treatment, relapses during treatment, or transforms into Richter. In the majority of cases, transformation evolves the original leukemia clone into a diffuse large B-cell lymphoma (DLBCL). Richter transformation (RT) represents a dreadful clinical challenge with limited therapeutic opportunities and scarce preclinical tools. CLL cells are well known to highly depend on survival signals provided by the tumor microenvironment (TME). These signals enhance the frequency of immunosuppressive cells with protumor function, including regulatory CD4+ T cells and tumor-associated macrophages. T cells, on the other hand, exhibit features of exhaustion and profound functional defects. Overall immune dysfunction and immunosuppression are common features of patients with CLL. The interaction between malignant cells and TME cells can occur during different phases of CLL development and transformation. A better understanding of in vivo CLL and RT biology and the availability of adequate mouse models that faithfully recapitulate the progression of CLL and RT within their microenvironments are “conditio sine qua non” to develop successful therapeutic strategies. In this review, we describe the xenograft and genetic-engineered mouse models of CLL and RT, how they helped to elucidate the pathophysiology of the disease progression and transformation, and how they have been and might be instrumental in developing innovative therapeutic approaches to finally eradicate these malignancies

The Interaction Between Two Worlds: MicroRNAs and Toll-Like Receptors

MicroRNAs (miRNAs) are critical mediators of posttranscriptional regulation via their targeting of the imperfect antisense complementary regions of coding and non-coding transcripts. Recently, researchers have shown that miRNAs play roles in many aspects of regulation of immune cell function by targeting of inflammation-associated genes, including Toll-like receptors (TLRs). Besides this indirect regulatory role of miRNAs, they can also act as physiological ligands of specific TLRs and initiate the signaling cascade of immune response. In this review, we summarize the potential roles of miRNAs in regulation of TLR gene expression and TLR signaling, with a focus on the ability of miRNAs bind to TLRs

Establishment and Characterization of PCL12, a Novel CD5+ Chronic Lymphocytic Leukaemia Cell Line.

Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents

(a) WB analysis of PCL12 cells, basal (-) and after IgM stimulation (+), for BCR signaling pathway proteins activation and expression.

<p>On the right a schematic representation of the BCR signaling pathway analyzed. (b) WB analysis after treatment with ibrutinib (5’, 30’, 1 hour, 24 hour) for BTK activation.(c) Transwell PCL12 migration assay of migration with and without SDF-1 stimulation. (d F-actin polymerization index on PCL12 cells in basal conditions and after IgM stimulation (e) confocal analysis of PCL12 cells stained for HS1 protein in green and DAPI in blue.</p

(a) Graph showing the subcutaneous tumor volume grow (b) CD19+ PCL12 cells infiltrating lymphoid organs quantified by flow cytometry in PB, SP, PE and BM.

<p>(c) Flow cytometric analysis of PCL12 cells in PB, surface CD19, CD5 and CD23 are represented. (d) images of the representative spleen from 1 mouse injected with PCL12 cells (1) and a WT (2). Histopathological examination of different tissues and IHC analysis.</p