Olga Frolova

Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

SDF-1alpha/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow-derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1alpha (SDF-1alpha)-induced and stroma-induced chemotaxis and inhibited SDF-1alpha-induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1alpha/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor- and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.

Regulation of HIF-1α signaling and chemoresistance in acute lymphocytic leukemia under hypoxic conditions of the bone marrow microenvironment

Overcoming resistance to chemotherapy is the main therapeutic challenge in the treatment of acute lymphocytic leukemia (ALL). Interactions between leukemia cells and the microenvironment promote leukemia cell survival and confer resistance to chemotherapy. Hypoxia is an integral component of bone marrow (BM) microenvironment. Hypoxia-inducible factor-1α (HIF-1), a key regulator of the cellular response to hypoxia, regulates cell growth and metabolic adaptation to hypoxia. HIF-1α expression, analyzed by Reverse Phase Protein Arrays in 92 specimens from newly diagnosed patients with pre-B-ALL, had a negative prognostic impact on survival (p = 0.0025). Inhibition of HIF-1α expression by locked mRNA antagonist (LNA) promoted chemosensitivity under hypoxic conditions, while pharmacological or genetic stabilization of HIF-1α under normoxia inhibited cell growth and reduced apoptosis induction by chemotherapeutic agents. Co-culture of pre-B ALL or REH cells with BM-derived mesenchymal stem cells (MSC) under hypoxia resulted in further induction of HIF-1α protein and acquisition of the glycolytic phenotype, in part via stroma-induced AKT/mTOR signaling. mTOR blockade with everolimus reduced HIF-1α expression, diminished glucose uptake and glycolytic rate and partially restored the chemosensitivity of ALL cells under hypoxia/stroma co-cultures. Hence, mTOR inhibition or blockade of HIF-1α-mediated signaling may play an important role in chemosensitization of ALL cells under hypoxic conditions of the BM microenvironment.

SL-401 and SL-501, targeted therapeutics directed at the interleukin-3 receptor, inhibit the growth of leukaemic cells and stem cells in advanced phase chronic myeloid leukaemia.

While imatinib and other tyrosine kinase inhibitors (TKIs) are highly efficacious in the treatment of chronic myeloid leukaemia (CML), some patients become refractory to these therapies. After confirming that interleukin‐3 receptor (IL3R, CD123) is highly expressed on CD34+/CD38− BCR‐ABL1+ CML stem cells, we investigated whether targeting IL3R with diphtheria toxin (DT)‐IL3 fusion proteins SL‐401 (DT388‐IL3) and SL‐501 (DT388‐IL3[K116W]) could eradicate these stem cells. SL‐401 and SL‐501 inhibited cell growth and induced apoptosis in the KBM5 cell line and its TKI‐resistant KBM5‐STI subline. Combinations of imatinib with these agents increased apoptosis in KBM5 and in primary CML cells. In six primary CML samples, including CML cells harbouring the ABL1 T315I mutation, SL‐401 and SL‐501 decreased the absolute numbers of viable CD34+/CD38−/CD123+ CML progenitor cells by inducing apoptosis. IL3‐targeting agents reduced clonogenic growth and diminished the fraction of primitive long‐term culture‐initiating cells in samples from patients with advanced phase CML that were resistant to TKIs or harboured an ABL1 mutation. Survival was also extended in a mouse model of primary TKI‐resistant CML blast crisis. These data suggest that the DT‐IL3 fusion proteins, SL‐401 and SL‐501, deplete CML stem cells and may increase the effectiveness of current CML treatment, which principally targets tumour bulk.

Ultraviolet irradiation induces multiple DNA double‐strand breaks and apoptosis in normal granulocytes and chronic myeloid leukaemia blasts

Major leucocyte subpopulations were isolated from peripheral blood of healthy donors, and patients with chronic myeloid leukaemia (CML) and chronic lymphoid leukaemia (CLL). In vitro UV irradiation was performed at the wavelength of 257 nm (UVC band). DNA double‐stranded breaks (DNAdsbs) were detected immediately after UV‐irradiation, by means of agarose gel electrophoresis. Cell viability was estimated after 18 h in culture, as relative numbers of residual non‐apoptotic cells. Evaluation of the dose–response curves revealed that normal CLL lymphoid cells showed only moderate damage after UV‐irradiation, as assessed by DNAdsbs and cell viability criteria. However, normal granulocytes and myeloid blasts from CML patients expressed a sharp increase in DNAdsbs, even at lower doses of UV‐radiation. UV‐induced amplification of endogenous oxidative systems (e.g. NADPH‐dependent oxidase) is suggested as a probable reason for enhanced DNA breakage and apoptosis in cells of the granulocytic lineage.