Ioanna Savvidou

Proliferation and bone marrow engraftment of AML blasts is dependent on β-catenin signalling.

B‐catenin is the central effector molecule of the canonical Wnt signalling pathway, which controls self‐renewal of haematopoietic stem cells. Deregulation of this pathway occurs in various malignancies including myeloid leukaemias. The present study examined the functional outcome of stable β‐catenin down‐regulation through lentivirus‐mediated expression of short hairpin RNA (shRNA). Reduction of the β‐catenin levels in acute myeloid leukaemia (AML) cell lines and patient samples decelerated their in vitro proliferation ability without affecting cell viability. Transplantation of leukaemic cells with control or reduced levels of β‐catenin in non‐obese diabetic severe combined immunodeficient animals indicated that, while the immediate homing of the cells was unaffected, the bone marrow engraftment was directly dependent on β‐catenin levels. Subsequent examination of bone sections revealed that β‐catenin was implicated in the localization of AML to the endosteum. Examination of adhesion molecule expression before and after transplantation, revealed down‐regulation of CD44 expression, accompanied by reduced in vitro adhesion. Gene expression analysis disclosed the presence of an autocrine compensatory mechanism, which responds to the reduced β‐catenin levels by altering the expression of positive and negative pathway regulators. In conclusion, our study showed that β‐catenin comprises an integral part of AML cell proliferation, cell cycle progression, and adhesion, and influences disease establishment in vivo.

Bioluminometric assay for relative quantification of mutant allele burden: application to the oncogenic somatic point mutation JAK2 V617F.

Unlike the inherited mutations, which are present in all cells, somatic (acquired) mutations occur only in certain cells of the body and, quite often, are oncogenic. Quantification of mutant allele burden (percentage of the mutant allele) is critical for diagnosis, monitoring of therapy, and detection of minimal residual disease. With point mutations, the challenge is to quantify the mutant allele while discriminating from a large excess of the normal allele that differs in a single base-pair. To this end, we report the first bioluminometric assay for quantification of the allele burden and its application to JAK2 V617F somatic point mutation, which is a recently (2005) discovered molecular marker for myeloproliferative neoplasms. The method is performed in microtiter wells and involves a single PCR, for amplification of both alleles, followed by primer extension reactions with allele-specific primers. The products are captured in microtiter wells and detected by oligo(dT)-conjugated photoprotein aequorin. The photoprotein is measured within seconds by simply adding Ca(2+). We have demonstrated that the percent (%) luminescence signal due to the mutant allele is linearly related to the allele burden. As low as 0.85% of mutant allele can be detected and the linearity extends to 100%. The assay is complete within 50 min after the amplification step.

β-Catenin Inhibitor BC2059 Is Efficacious as Monotherapy or in Combination with Proteasome Inhibitor Bortezomib in Multiple Myeloma

Currently available treatment options are unlikely to be curative for the majority of multiple myeloma patients, emphasizing a continuing role for the introduction of investigational agents that can overcome drug resistance. The canonical Wnt/β-catenin signaling pathway, essential for self-renewal, growth, and survival, has been found to be dysregulated in multiple myeloma, particularly in advanced stages of disease. This provides the rationale for evaluating the novel β-catenin inhibitor BC2059 as monotherapy and in combination with proteasome inhibitors in vitro and in vivo. Here, we show nuclear localization of β-catenin in human myeloma cell lines (HMCL), consistent with activation of the canonical Wnt pathway. BC2059 attenuates β-catenin levels, in both the cytoplasm and the nucleus, reducing the transcriptional activity of the TCF4/LEF complex and the expression of its target gene axin 2. Treatment of HMCL with BC2059 inhibits proliferation and induces apoptosis in a dose-dependent manner. This is also observed in HMCL–stromal cell cocultures, mitigating the protective effect afforded by the stroma. Similarly, BC2059 induces apoptosis in primary multiple myeloma samples in vitro, causing minimal apoptosis on healthy peripheral blood mononuclear cells. Furthermore, it synergizes with the proteasome inhibitor bortezomib both in HMCL and primary multiple myeloma samples. Finally, in xenograft models of human myelomatosis, BC2059 delays tumor growth and prolongs survival with minor on-target side effects. Collectively, these results demonstrate the efficacy of targeting the Wnt/β-catenin pathway with BC2059 both in vitro and in vivo, at clinically achievable doses. These findings support further clinical evaluation of BC2059 for the treatment of multiple myeloma. Mol Cancer Ther; 16(9); 1765–78. ©2017 AACR.

Intravascular B-cell lymphoma with leukemic presentation: case report and literature review

Intravascular lymphoma is an extremely rare, disseminated, and aggressive extranodal CD20+ non‐Hodgkin’s lymphoma characterized by the presence of lymphoma cells only in the lumina of small vessels. We report a 72‐year‐old woman with a diagnosis of intravascular lymphoma presented with splenomegaly and leukemic appearance in the peripheral blood smear. Her clinical course was rapidly deteriorated before the initiation of specific chemotherapy and finally died due to multiorgan insufficiency. Bone marrow biopsy revealed a characteristic infiltration of CD5, CD10 B‐cell lymphoma. To our knowledge, this is the first reported case of a CD5, CD10 intravascular B‐cell lymphoma with leukemic presentation in peripheral blood with multiple cytogenetic aberrations.