CD44 regulates the apoptotic response and promotes disease development in chronic lymphocytic leukemia.

The cell-surface glycoprotein CD44 is expressed in chronic lymphocytic leukemia (CLL), but its functional role in this disease is poorly characterized. We therefore investigated the contribution of CD44 to CLL in a murine disease model, the Eµ-TCL1 transgenic mouse, and in CLL patients. Surface CD44 increased during murine CLL development. CD44 expression in human CLL was induced by stimulation with interleukin 4/soluble CD40 ligand and by stroma cell contact. Engagement of CD44 by its natural ligands, hyaluronic acid or chondroitin sulfate, protected CLL cells from apoptosis, while anti-CD44 small interfering RNAs impaired tumor cell viability. Deletion of CD44 during TCL1-driven murine leukemogenesis reduced the tumor burden in peripheral blood and spleen and led to a prolonged overall survival. The leukemic cells from these CD44 knockout animals revealed lower levels of antiapoptotic MCL1, a higher propensity to apoptosis, and a diminished B-cell receptor kinase response. The inhibitory anti-CD44 antibodies IM7 and A3D8 impaired the viability of CLL cells in suspension cultures, in stroma contact models, and in vivo via MCL1 reduction and by effector caspase activation. Taken together, CD44 expression in CLL is mediated by the tumor microenvironment. As a coreceptor, CD44 promotes leukemogenesis by regulating stimuli of MCL1 expression. Moreover, CD44 can be addressed therapeutically in CLL by specific antibodies.

Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34(+) peripheral blood progenitor cells.

BACKGROUND AND AIMS: Because of their pluripotency, human CD34(+) peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC. METHODS: A panel of pseudotyped AAV vectors (designated AAV2/x, containing the vector genome of serotype 2 and capsid of serotype x, AAV2/1-AAV2/6) was screened on primary human granulocyte-colony-stimulating factor (G-CSF)-mobilized CD34(+) PBPC to determine their gene transfer efficacy. Additionally, double-stranded self-complementary AAV (dsAAV) were used to determine possible second-strand synthesis limitations. RESULTS: AAV2/6 vectors proved to be the most efficient [12.8% (1.8-25.4%) transgene-expressing PBPC after a single transduction], being significantly more efficient (all P<0.005) than the other vectors [AAV2/2, 2.0% (0.2-7.3%); AAV2/1, 1.3% (0.1-2.9%); others, <; 1% transgene-expressing PBPC]. In addition, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using pseudotyped dsAAV vectors: for dsAAV2/2 [9.3% (8.3-20.3%); P<0.001] and dsAAV2/6 [37.7% (23.6-61.0%); P<0.001) significantly more PBPC expressed the transgene compared with their single-stranded counterparts; for dsAAV2/1, no significant increase could be observed. CONCLUSIONS: We have shown that clinically relevant transduction efficiency levels using AAV-based vectors in human CD34(+) PBPC are feasible, thereby offering an efficient alternative vector system for gene transfer into this important target cell population.

Generation of efficient human blood progenitor-targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells.

OBJECTIVE: Currently standard recombinant adeno-associated virus serotype 2(rAAV2)-based vectors lack the efficiency for gene transfer into primary human CD34(+) peripheral blood progenitor cells (PBPC). MATERIALS AND METHODS: An advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34(+) PBPC cells. RESULTS: On a panel of leukemia cell lines (CML/AML), significantly higher gene transfer efficiency of the rAAV capsid mutants (up to 100% gene transfer) was observed compared to standard rAAV2 vectors. A higher transduction efficiency in the imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S and a pronounced difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S were particularly striking. On solid tumor cell lines, on the other hand, rAAV2 was more efficient than the capsid mutants, suggesting an increased specificity of our capsid mutants for hematopoietic progenitor cells. On primary human CD34(+) PBPC significantly higher (up to eightfold; 16% green fluorescent protein-positive) gene transfer could be obtained with the newly generated vectors compared to standard rAAV2 vectors. CONCLUSION: These novel vectors may enable efficient gene transfer using rAAV-based vectors into primary human blood progenitor cells for a future clinical application.

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line.

BACKGROUND: For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. METHODS: To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. RESULTS: Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% +/- 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% +/- 2% GFP+ cells; Lama84: 36-fold, 29% +/- 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% +/- 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% +/- 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. CONCLUSION: Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors.

Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia.

Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.