Julie Turzanski

P-glycoprotein in acute myeloid leukaemia: Therapeutic implications of its association with both a multidrug-resistant and an apoptosis-resistant phenotype

P-glycoprotein (Pgp) expression is an independent prognostic factor for response to remission-induction chemotherapy in acute myeloblastic leukaemia, particularly in the elderly. There are several potential agents for modulating Pgp-mediated multi-drug resistance, such as cyclosporin A and PSC833, which are currently being evaluated in clinical trials. An alternative therapeutic strategy is to increase the use of drugs which are unaffected by Pgp. However, in this review, we explain why this may be more difficult than it appears. Evidence from in vitro studies of primary AML blasts supports the commonly held supposition that chemoresistance may be linked to apoptosis-resistance. We have found that Pgp has a drug-independent role in the inhibition of in vitro apoptosis in AML blasts. Modulation of cytokine efflux, signalling lipids and intracellular pH has all been suggested as ways by which Pgp may affect cellular resistance to apoptosis include modulation of cytokine efflux, signalling lipids and intracellular pH; these are discussed in this review. For a chemosensitising agent to be successful, it may be more important for it to enhance apoptosis than to increase drug uptake.

Use of standardized flow cytometric determinants of multidrug resistance to analyse response to remission induction chemotherapy in patients with acute myeloblastic leukaemia

We have used a combination of flow cytometric assays to define multidrug resistance (MDR) positive and negative blasts in cryopreserved samples from 47 MRC trial patients with acute myeloblastic leukaemia (AML). Our primary test is a standardized assay for daunorubicin accumulation. Confirmatory assays for MDR comprised the cyclosporin modulation assay for rhodamine‐123 uptake as a measure of functional P‐glycoprotein and the measurement of lung resistance protein and multidrug resistance associated protein (with LRP‐56 and MRPr1 respectively).

Early allogeneic transplantation for refractory or relapsed acute leukaemia following remission induction with FLAG

The prognosis for patients with secondary AML, primary resistant AML or ALL and early (<12 months) relapse of acute leukaemia remains extremely poor with conventional chemotherapy. as part of a strategy to improve the outcome for these patients we have treated 22 consecutive patients (18 aml, four all, median age 35 years) with either primary resistant disease (n = 3), early relapsed leukaemia (n = 12) or secondary AML (n = 7, four RAEBt, two antecedant ALL and one antecedant Hodgkin’s disease) with ‘FLAG’ induction chemotherapy with the aim of proceeding to early allogeneic transplantation either from sibling or unrelated donors. Eighteen patients achieved CR after one course of FLAG, including five patients who had documented p-glycoprotein-induced multidrug resistance and 10 patients with adverse cytogenetic abnormalities. Eight patients were consolidated with a second course of FLAG prior to transplantation and so far 16 patients have undergone allogeneic transplantation, 10 from unrelated donors and six from sibling donors (one mismatched). By the time of transplant three patients had progressed and were in early relapse and all have relapsed post BMT. Of the remaining 13 patients transplanted in remission, nine remain in CCR at a range of 4–26 months, three have died of transplant-related complications (18%) and one patient has relapsed. We conclude that the use of FLAG induction therapy followed by early allogeneic transplantation from either a sibling or unrelated donor can be an effective strategy for the treatment of this difficult group of young patients with poor risk acute leukaemia and appears to be associated with a low procedure-related risk.

Resistance to spontaneous apoptosis in acute myeloid leukaemia blasts is associated with p-glycoprotein expression and function, but not with the presence of FLT3 internal tandem duplications

Summary. The ability of acute myeloid leukaemia (AML) blasts to survive in culture has been associated with poor patient response to chemotherapy. Other biological factors predicting an adverse outcome include p‐glycoprotein (pgp) expression, which is associated with a reduced remission rate, and the presence of fms‐like tyrosine kinase 3 gene (FLT3) internal tandem duplications (ITDs), predictive of a high rate of leukaemic relapse. Our previous work has indicated a drug efflux‐independent role for pgp in apoptosis resistance. We measured spontaneous in vitro apoptosis in 58 primary AML samples to establish its relationship with functional and phenotypic pgp and with FLT3 ITDs. Cells were incubated for 48 h in a suspension culture, and the remaining viable cells were counted by flow cytometry. Median survival was 38% of baseline values. Resistance to spontaneous apoptosis was strongly associated with pgp (MRK‐16 antibody) expression (P = 0·001) and with pgp functional activity (P < 0·001). FLT3 ITDs, found in 20 cases, were inversely associated with functional pgp activity: thus, the median pgp modulation ratio was 2·0 in FLT3 wild‐type cases and 1·38 in ITD cases (P = 0·018). Also, the presence of FLT3 ITDs was not associated with in vitro apoptosis resistance. In conclusion, we have found that the presence of FLT3 ITDs is not related to AML blast survival in vitro, and is inversely associated with pgp activity, whereas pgp expression and activity are associated with resistance to spontaneous apoptosis. These results may help to explain the differing adverse effects of pgp (on remission induction) and FLT3 ITDs (on relapse) in AML.

Interleukin-1β maintains an apoptosis-resistant phenotype in the blast cells of acute myeloid leukaemia via multiple pathways

Blast cell survival in suspension culture is associated with chemoresistance in acute myeloid leukaemia (AML). Autonomous production of IL-1β by AML blasts is linked with a proliferative response, although its role in survival and hence apoptosis-resistance has not been examined in this disease. Cells that secreted more than 19.7 pg/ml IL-1β were significantly more resistant to spontaneous apoptosis in 48-h culture than those that produced less than 19.7 pg/ml IL-1β (P=0.008). Exogenous rhIL-1β significantly enhanced 48-h survival in 25/29 blast cell samples (P=0.0001). IL-1 receptor ligation is known to activate at least three survival pathways: those mediated by PI-3 kinase, IL-1 receptor-associated kinase (IRAK) and ceramidase. In apoptosis-sensitive AML blasts with a strong survival response to rhIL-1β, inhibitors of all three pathways down-modulated an IL-1β-mediated increase in blast survival, but only the inhibition of all three pathways totally eliminated viable blasts. In apoptosis-resistant and apoptosis-sensitive primary AML samples, the three inhibitors all increased apoptosis in vitro after 48 h. Exogenous rhIL-1β induced the hyperphosphorylation of Bcl-2. It also increased the activation of NF-κB in 5/15 blast samples. IL-1β-mediated survival pathways may be a factor in apoptosis-resistance in primary AML blasts, and may therefore contribute to chemoresistance.

Contrasting in vitro effects for the combination of fludarabine, cytosine arabinoside (Ara-C) and granulocyte colony-stimulating factor (FLAG) compared with daunorubicin and Ara-C in P-glycoprotein-positive and P-glycoprotein-negative acute myeloblastic leukaemia

It has been suggested that the FLAG remission induction regimen comprising fludarabine (F‐ara), cytosine arabinoside (Ara‐C) and granulocyte colony‐stimulating factor (G‐CSF) may be capable of overcoming P‐glycoprotein (P‐gp)‐related multidrug resistance (MDR) in patients with acute myeloblastic leukaemia (AML). We have investigated the in vitro response of P‐gp‐positive and ‐negative AML clones to FLAG and compared this with their response to treatment with Ara‐C and daunorubicin (DNR). Twenty‐four cryopreserved samples from patients with AML were studied using a flow cytometric technique for the enumeration of viable (7‐amino actinomycin D negative) cells. Samples consisted of 12 P‐gp‐positive and 12 P‐gp‐negative cases, as measured by the MRK16 antibody. The results were analysed by calculating the comparative drug resistance (CDR), i.e. the percentage cell death caused by Ara‐C + DNR subtracted from the percentage cell death, caused by FLAG after 48 h incubation in suspension culture. P‐gp‐positive clones were shown to have a significantly higher CDR than P‐gp‐negative clones (P = 0·001). Furthermore, a significant positive correlation (r2 = 0·40, P < 0·01) was found between P‐gp protein expression and CDR. However, P‐gp function, measured using cyclosporin modulation of rhodamine 123 (R123) uptake, was not associated with the CDR, demonstrating that there are other properties of P‐gp, besides its role in drug efflux, that modulate the responsiveness of AML blasts to chemotherapy. These results are consistent with a potential benefit for FLAG in P‐gp‐positive AML, but not P‐gp‐negative AML, compared with standard anthracycline and Ara‐C therapy.

Blast maturity and CD34 expression determine the effects of the differentiating agents KH1060 and 9-cis-retinoic acid on the differentiation and clonogenicity of non-M3 acute myeloid leukaemia cells

The vitamin D analogue KH1060 and the retinoids all-trans retinoic acid (ATRA), 9-cis-retinoic acid (9-cRA) and 4-hydroxyphenyl retinamide (4-HPR) induce differentiation and/or apoptosis and inhibit clonal growth of acute promyelocytic leukaemia cells. We have studied the effects of these agents in vitro on cells from 12 patients with other forms of acute myeloblastic leukaemia (AML). Treatment with KH1060 (10−6 M) caused decreases in cell viability in suspension culture to a median of 44% of control values (P = 0.02). However, retinoids had little effect. Subsequent clonal growth in semi-solid medium was inhibited to 5% (median) of control with 10−6 M KH 1060 (P = 0.03) and to 73% with 10−6 M 9-cRA (P = 0.01). Further inhibition of clonal growth by the combination of 5 × 10−7 M 9-cRA and 5 × 10−7 M KH1060 was only noted in one case. Following the primary suspension culture, cells from 6/6 CD34 positive samples grew in semi-solid cultures without analogues, whereas cells from 3/6 CD34 negative cultures grew. 10−6 M KH1060 completely abolished colony growth in all three CD34 negative samples and 10−6 M 9-cRA inhibited the number of colonies to a median of 11% of control values. In the six CD34 positive samples median colony growth was inhibited to 36% of control values by KH1060 and to 83% of control values by 9-cRA. CD11b expression was increased by 210% (median) with 9-cRA and by 90% (median) with KH1060 in early to intermediate myeloblast (M0, M1, M2) clones. A different pattern was noted in more mature (M4, M5, M6) clones: here there was little or no increase in CD11b expression induced by retinoids or KH1060, but the ratio of apoptotic to viable CD11b+ cells, measured by CD11b/7-AAD double staining, was increased in 6/6 cases treated with KH1060 or the combination of 9-cRA and KH 1060, and in 5/6 cases treated with 9-cRA. No overall significant change in bcl-2 or bax expression on G0/G1 cells was found after 3 days’ suspension culture with the analogues. However bcl-x was downregulated in G0/G1 cells treated with KH1060 (median bcl-x relative fluorescence intensity = 45.3 in cells treated with KH1060, compared with 65.7 in control wells, P = 0.028). We conclude that CD34+ samples are relatively resistant to the growth inhibition induced by KH1060 and 9-cRA. However, downregulation of bcl-x in cells which have survived treatment with KH1060 may increase the susceptibility of the remaining leukaemic cells to cytotoxic drugs.