P Dello Sbarba

The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability

In liquid cultures of murine bone marrow cells stimulated with interleukin-3 and granulocyte/macrophage colony-stimulating factor, hypoxia (1% oxygen) induced a reversible block of hematopoiesis, maintaining the progenitors’ expansion potential unreduced. Progenitors repopulating day-14 hypoxic cultures with cells or granulocyte/macrophage colony-forming units (CFU-GM) were found, on the basis of their maintenance in hypoxia (12% and 76%, respectively), to belong to different subsets, the latter being much more efficiently maintained. The maintenance in hypoxic cultures of progenitors detectable by marrow-repopulating ability (MRA) assay was 18% for MRAcell progenitors and 69% for MRACFU progenitors. Thus, the repopulation of hypoxic cultures with cells or CFU-GM closely reflected the presence of progenitors capable of repopulating, with cells or CFU-GM, the bone marrow of lethally irradiated syngeneic animals. Progenitors repopulating hypoxic cultures were, like MRA progenitors, significantly resistant to 5- fluorouracil, progenitors repopulating cultures with CFU-GM being two-fold more resistant than those repopulating cultures with cells. We concluded that the repopulation of day-14 hypoxic cultures occurring after their transfer to air is to be considered an indicator of the maintenance of MRA progenitors in hypoxia. The relevance of these results to stem cell biology and their potential practical applications are discussed.

Hypoxia suppresses BCR/Abl and selects imatinib-insensitive progenitors within clonal CML populations.

Hypoxia suppresses BCR/Abl and selects imatinib-insensitive progenitors within clonal CML populations

Selective anti-leukaemic activity of low-dose histone deacetylase inhibitor ITF2357 on AML1/ETO-positive cells.

We analysed the in vitro effects of a new hydroxamate derivative, ITF2357, on AML cells. ITF2357 potently induced histone acetylation. ITF2357 0.1 μM blocked proliferation and induced apoptosis in AML1/ETO-positive Kasumi-1 cells, while AML1/ETO-negative HL60, THP1 and NB4 cell lines were sensitive only to 1 μM ITF2357. Apoptosis was induced by 0.1 μM ITF2357 in AML1/ETO-positive primary blasts and U937-A/E cells induced to express AML1/ETO, but not in U937-A/E cells non-expressing AML1/ETO. In Kasumi-1 cells 0.1 μM ITF2357 induced AML1/ETO degradation through a caspase-dependent mechanism. ITF2357 0.1 μM also determined DNMT1 efflux from, and p300 influx to, the nucleus. Moreover, 0.1 μM ITF2357 determined local H4 acetylation and release of DNMT1, HDAC1 and AML1/ETO, paralleled by recruitment of p300 to the IL-3 gene promoter. ITF2357 treatment, however, did not induce re-expression of IL-3 gene. Accordingly, the methylation level of IL-3 promoter, as well as of several other genes, was unmodified. In conclusion, ITF2357 emerged as an anti-leukaemic agent very potent on AML cells, and on AML1/ETO-positive cells in particular. More relevantly, clearly emerged from our results that ITF2357 could be an ideal agent to treat AML subtypes presenting AML1/ETO fusion protein which determine HDAC involvement in leukaemogenesis.

AML1/ETO sensitizes via TRAIL acute myeloid leukemia cells to the pro-apoptotic effects of hypoxia.

We determined the effects of severe hypoxia (∼0.1% O2) on acute myeloid leukemia cells expressing the AML1/ETO oncogene. Incubation of Kasumi-1 cells in hypoxia induced growth arrest, apoptosis and reduction of AML1/ETO protein expression. The conditional expression of AML1/ETO in U937-A/E cells showed that hypoxia induces marked apoptosis in AML1/ETO-expressing cells only, pointing to AML1/ETO as a factor predisposing cells to hypoxia-induced apoptosis. In AML1/ETO-expressing cells, hypoxia enhanced TRAIL expression and its proapoptotic effects. AML1/ETO was found to bind TRAIL promoter and induce TRAIL transcription, although TRAIL expression was restrained by a concomitant relative transcription block. In hypoxia, such a TRAIL repression was removed and an increase of TRAIL expression was induced. Finally, blocking anti-TRAIL antibodies markedly reduced (Kasumi-1 cells) or completely inhibited (U937-A/E cells) hypoxia-induced apoptosis. Taken together, these results indicated that hypoxia induces apoptosis in AML1/ETO-expressing cells via a TRAIL/caspase 8-dependent autocrine loop and that TRAIL is a key regulator of hypoxia-induced apoptosis in these cells.

Salarin C inhibits the maintenance of chronic myeloid leukemia progenitor cells

We previously showed that incubation of chronic myeloid leukemia (CML) cells in very low oxygen selects a cell subset where the oncogenetic BCR/Abl protein is suppressed and which is thereby refractory to tyrosine kinase inhibitors used for CML therapy. In this study, salarin C, an anticancer macrolide extracted from the Fascaplysinopsis sponge, was tested as for its activity on CML cells, especially after their incubation in atmosphere at 0.1% oxygen. Salarin C induced mitotic cycle arrest, apoptosis and DNA damage. Salarin C also concentration-dependently inhibited the maintenance of stem cell potential in cultures in low oxygen of either CML cell lines or primary cells. Surprisingly, the drug also concentration-dependently enforced the maintenance of BCR/Abl signaling in low oxygen, an effect which was paralleled by the rescue of sensitivity of stem cell potential to IM. These results suggest a potential use of salarin C for the suppression of CML cells refractory to tyrosine kinase inhibitors

A comparative kinetic analysis of proliferation in vitro of Con‐A‐treated splenocytes and syngeneic leukaemia cells

Abstract. The growth kinetics of Con‐A‐treated mouse splenocytes and syngeneic leukaemia cells cultured in vitro were compared with respect to (i) the total cell number, (ii) the rate of [14C]thymidine incorporation (measured by pulse‐labelling the cells at various times of incubation), and (iii) the labelling index of the cell populations. By correlating the thymidine incorporation, labelling index and cell number data, it has been established that, for both types of cells, the rate of [14C]thymidine incorporation is directly proportional to the number of cells synthesizing DNA. A new approach to cytokinetic analysis has been developed, showing that important information can be obtained by determining the cumulative kinetics of [14C]thymidine incorporation. The latter has been calculated by integrating the area underneath the time course of the rate of thymidine incorporation, and was directly proportional to the overall growth of both leukaemia cells and Con‐A‐stimulated splenocytes. Based on this proportionality, an estimate of the average duration of the S phase for both types of cells was calculated, suggesting that normal and neoplastic blasts maintain this parameter at a constant value (7.6 and 5.9 hr, respectively) throughout different stages of growth. The percentage of Con‐A‐responsive cells within the initial splenocyte population and their overall proliferation in vitro have been determined by a procedure which measures the cumulative kinetics of thymidine incorporation and the kinetics of cell total number in the presence or in the absence of the lectin, as well as in the presence of Con‐A plus colcemid. A minor fraction (11%) of the initial splenocytes is recruited into cycle by Con‐A, proliferating with similar kinetics to that of leukaemia cells in the same conditions. The great majority of the initial splenocyte population is unaffected by Con‐A, decaying exponentially throughout the incubation with the same half‐life (28 hr), both in the presence or in the absence of the lectin.

PO-145 ERK5 pathway inhibitors inhibit the maintenance of chronic myeloid leukaemia stem cells

Introduction Chronic myeloid leukaemia (CML) is a hematopoietic stem cell (HSC)-driven neoplasia characterised by the expression of the constitutively active tyrosine kinase BCR/ABL. CML therapy based on tyrosine kinase inhibitors (TKi) is highly effective in inducing remission but not in targeting leukaemia stem cells (LSC), which sustain the minimal residual disease and are responsible for CML relapse following discontinuation of treatment. Our aim was to address the effects of the inhibition of the ERK5 pathway on the maintenance of CML LSC. Material and methods KCL22 and K562 CML cell lines, patient-derived CML cells or CD34 +peripheral blood cells from healthy donors (informed consent) were incubated in normoxic or hypoxic (0.1% O2) primary cultures (LC1) in the presence or the absence of drugs. At the end of incubation (day 7), cells were analysed on a flow cytometer to determine the expression of stem cell markers or transferred to drug-free normoxic secondary cultures (LC2) to measure LC2 repopulation as a read-out of progenitor/stem cell potential (CRA assay). In the serial Colony Formation Ability (CFA) assay colonies were scored on day 7 of each passage (III passages). In the Long-Term Culture-Initiating Cells (LTC-IC) assay the number of colonies was scored after 14 days. Compounds: XMD8-92 (ERK5 inhibitor) and BIX02189 (MEK5 inhibitor); imatinib and dasatinib (BCR/ABL inhibitors). Results and discussions In CML patient-derived cells and cell lines, we found that the MEK5/ERK5 pathway is active and necessary for optimal proliferation in low oxygen, a condition typical of normal hematopoietic and leukemic stem cell niches. Treatment of primary CML cells with XMD8-92 or BIX02189, but not with TKi, strikingly reduced Culture Repopulation Ability (CRA), serial Colony Formation Ability and Long-Term Culture-Initiating Cells (LTC-IC). Importantly, inhibition of MEK5/ERK5 was effective on CML cells regardless of the presence or absence of imatinib (IM), and did not reduce CRA or LTC-IC of normal CD34 +cells. Interestingly, in hypoxia, combined treatment XMD8-92/IM decreased the expression of genes relevant for stem cell maintenance such as c-MYC, SOX2 and NANOG and the expression of CD26, a CML LSC marker. Conclusion We propose ERK5 pathway inhibitors as a novel therapeutic approach to prevent CML relapse and, in combination with TKi, enhance induction of remission.