alitretinoin and Leukemia--Promyelocytic--Acute

Despite achieving complete remission with retinoic acid (RA), most patients with acute promyelocytic leukemia (APL) have minimal residual disease detectable by reverse transcription-PCR (RT-PCR) amplification. HuM195, a humanized monoclonal antibody reactive with the cell surface antigen CD33, specifically targets and kills myeloid leukemia cells. We studied whether HuM195 could eliminate minimal residual disease in patients with APL by using RT-PCR. After attaining clinical complete remission with RA and/or chemotherapy, patients received HuM195 twice weekly for 3 weeks. Patients in first remission were given consolidation chemotherapy, generally with three cycles of idarubicin and cytarabine. Patients in second or greater remission did not receive chemotherapy. All patients received six monthly courses of maintenance with two doses of HuM195. Twenty-five of 27 patients treated in first remission had positive RT-PCR determinations before HuM195 treatment. Of the 22 patients evaluable for conversion of positive RT-PCR assays, 11 (50%) became RT-PCR negative after HuM195 treatment without additional therapy. Within the subset of patients who received RA alone as induction, 8 of 18 evaluable patients (44%) had negative RT-PCR determinations after HuM195 treatment but before chemotherapy. Among similar patients treated on earlier studies, 7 of 34 patients (21%) induced into remission with RA and then maintained on the drug for 1 month were RT-PCR negative before chemotherapy (P = 0.07). Twenty-five of 27 patients with newly diagnosed APL (93%) remain in clinical complete remission for 7+ to 58+ months, with median follow-up of 29 months. Seven patients in second or third remission and one patient in molecular relapse were also treated. Only one of these patients became RT-PCR negative after treatment with HuM195. These data suggest that HuM195 has activity against minimal residual disease in APL, particularly in newly diagnosed patients.

Topics: Adolescent; Adult; Aged; Alitretinoin; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antineoplastic Agents; Disease-Free Survival; Female; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Remission Induction; Sialic Acid Binding Ig-like Lectin 3; Time Factors; Tretinoin

The use of all-trans retinoic acid (RA) for remission induction markedly increases survival of patients with acute promyelocytic leukemia (APL) compared to patients treated solely with cytotoxic chemotherapy. However, clinical resistance to this agent develops rapidly, which has been associated with a progressive decline in plasma drug concentrations. Previous studies suggested that 9-cis RA, a retinoid receptor 'pan agonist' did not induce its own catabolism to the same extent as all-trans RA. Therefore, we conducted a dose-ranging study of this compound in patients with both relapsed and newly diagnosed APL. We treated 18 patients with morphologically diagnosed APL (13 relapsed, five newly diagnosed). The daily dose of 9-cis RA ranged from 30 to 230 mg/m2/day given as a single oral dose. Four of 12 (33%) relapsed patients (three of whom were previously treated with all-trans RA) and four of five (80%) newly diagnosed patients achieved complete remission. The sole failure in the newly diagnosed group died early from an intracranial hemorrhage. One other patient with t(9;12) translocation had substantial hematologic improvement. The drug was generally well tolerated; headache and dry skin were the most common adverse reactions. Three patients were treated with corticosteroids for signs of incipient 'RA syndrome.' These preliminary data suggest that 9-cis RA is an effective agent for remission induction and deserves further investigation in patients with retinoid-sensitive APL.

Topics: Adolescent; Adult; Aged; Alitretinoin; Antineoplastic Agents; Child; Child, Preschool; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 9; Dose-Response Relationship, Drug; Female; Humans; Infant; Leukemia, Promyelocytic, Acute; Leukocyte Count; Male; Middle Aged; Platelet Count; Recurrence; Translocation, Genetic; Tretinoin

New structure-activity relationships of a series of methylene or side chain modified retinoids on NB4 acute promyelocytic leukemia cells are investigated. The differentiation- and apoptosis-inducing potential of these compounds is analyzed on the basis of their selective retinoic acid receptor binding profile.

Topics: Cell Death; Cell Differentiation; Cell Line, Tumor; Humans; Hydrocarbons; Leukemia, Promyelocytic, Acute; Methane; Retinoids; Structure-Activity Relationship

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Benzoates; Caspases; Cell Differentiation; Cell Division; Enzyme Activation; Gene Expression; Humans; Leukemia, Promyelocytic, Acute; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured

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 x 10(-7) M 9-cRA and 5 x 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.

Topics: Alitretinoin; Antigens, CD34; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Calcitriol; Cell Differentiation; Cell Division; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured

The EVI-1 gene encodes a Zn finger, DNA binding protein previously detected in some acute myelogenous leukemias (AML) and myelodysplasias (MDS), but not in normal marrow or cord blood cells. Experimental studies suggest EVI-1 blocks cellular differentiation by binding to GATA-1 or other specific DNA sequences controlling gene expression, and may be involved in the pathogenesis of some AMLs. To further define potential roles for EVI-1 in leukemia pathogenesis, we studied its regulation in acute promyelocytic leukemias (APL). Seven of 11 APL cases expressed EVI-1 RNA detected by RNA PCR at diagnosis, and expression was detected in two additional cases after treatment with all-trans retinoic acid (ATRA). Two of four cases studied at relapse also expressed EVI-1 RNA. To investigate regulation of EVI-1 expression in APL, we examined its expression in the NB4 APL cell line. NB4 cells did not express EVI-1 under basal conditions, but expressed EVI-1 after ATRA-induced differentiation. When NB4 cells were exposed to ATRA and transferred to cultures with N,N'-hexamethylene-bis-acetamide (HMBA), differentiation occurred but EVI-1 RNA was not detected, indicating that EVI-1 expression was not required for terminal, NB4 differentiation. ATRA-resistant NB4 cells were obtained by continuous culture in gradually increasing concentrations of ATRA. These cells did not express markers of differentiation but continued to express EVI-1 for several weeks even after ATRA withdrawal. To assess whether expression of the APL PML-RAR alpha fusion gene alone was sufficient for ATRA induction of EVI-1, the PML-RAR alpha gene cDNA was expressed in U937 histiocytic lymphoma cells. ATRA treatment of PML-RAR alpha-transfected or control U937 cells did not induce EVI-1 expression. In conclusion, this study demonstrates the EVI-1 gene is consistently expressed in APL cells either constitutively or after ATRA treatment. ATRA represents the first biologically active agent shown to specifically regulate EVI-1 expression in blood cells. In contrast to previous studies in AML and MDS, the pattern of EVI-1 expression suggests it may facilitate rather than inhibit myeloid differentiation during ATRA treatment. However, effects of EVI-1 expression are likely to be complex, and expression in ATRA-resistant APL cells may indicate multiple roles for this gene.

Topics: Acetamides; Alitretinoin; Cell Differentiation; DNA-Binding Proteins; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Isotretinoin; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; MDS1 and EVI1 Complex Locus Protein; Neoplasm Proteins; Oncogene Proteins, Fusion; Proto-Oncogenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Zinc Fingers

The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha) are the two major fusion proteins expressed in acute promyelocytic leukemia (APL) patients. These proteins, which are present in different lengths of PML (amino acids 1-552 and 1-394, respectively), contain most of the functional domains of PML and RAR alpha, bind all-trans-retinoic acid (t-RA), and act as t-RA-dependent transcription factors. T-RA is an effective inducer of clinical remission only in patients carrying the t(15;17) and expressing the PML/RAR alpha products. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RAR alpha product has been found associated with a poorer prognosis than bcr1-PML/RAR alpha. In the present study we have investigated the structural and functional properties of the bcr3-PML/RAR alpha in comparison to the previously characterized bcr1-PML/RAR alpha. In particular, we have measured the binding properties of the two endogenous ligands t-RA and 9-cis-RA to both of these isoforms. T-RA binding analysis of nuclear and cytosolic extracts prepared from bcr3-PML/RAR alpha APL patients and from bcr3-PML/RAR alpha COS-1 transfected cells indicates that this protein is present only as high-molecular-weight nuclear complexes. Using saturation binding assays and Scatchard analyses we found that t-RA binds with slightly less affinity to the bcr3-PML/RAR alpha receptor than to bcr1-PML/RAR alpha or RAR alpha (Kd = 0.4 nmol/L, 0.13 nmol/L or 0.09 nmol/L, respectively). Moreover, two different high-affinity 9-cis-RA binding sites (Kd = 0.45 and 0.075 nmol/L) were detectable in the bcr3-PML/RAR alpha product but not in the bcr1-PML/RAR alpha product (Kd = 0.77 nmol/L). By competition binding experiments we showed that 9-cis-RA binds with higher specificity to the bcr3-PML/RAR alpha isoform than to the bcr1-PML/RAR alpha or RAR alpha. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RAR alpha product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the betaRARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RAR alpha products can be measured.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Binding, Competitive; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; COS Cells; DNA-Binding Proteins; Gene Expression Regulation, Leukemic; Humans; Kruppel-Like Transcription Factors; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Prognosis; Promyelocytic Leukemia Zinc Finger Protein; Protein Binding; Recombinant Fusion Proteins; Remission Induction; Structure-Activity Relationship; Transcription Factors; Transcription, Genetic; Transfection; Translocation, Genetic; Tretinoin

Two types of aromatic amides, terephthalic monoanilides and (arylcarboxamido)benzoic acids, have been shown to possess potent retinoidal activities and can be classified as retinoids. The structure-activity relationships of these amides are discussed on the basis of differentiation-inducing activity on human promyelocytic leukemia cells HL-60. In generic formula 4 (X = NHCO or CONH), the necessary factors to elicit the retinoidal activities are a medium-sized alkyl group (isopropyl, tert-butyl, etc.) at the meta position and a carboxyl group at the para position of the other benzene ring. The bonding of the amide structure can be reversed, this moiety apparently having the role of locating the two benzene rings at suitable positions with respect to each other. Substitution at the ring position ortho to the amide group or N-methylation of the amide group caused loss of activity, presumably owing to the resultant change of conformation. It is clear that the mutual orientation of the benzylic methyl group(s) and the carboxyl group and their distance apart are essential factors determining the retinoidal activity. Among the synthesized compounds, 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benz oic acid (Am80) and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido] benzoic acid (Am580) were several times more active than retinoic acid in the assay. They are structurally related to retinoic acid, as is clear from the biological activity of the hybrid compounds (M2 and R2).

Topics: Amides; Binding Sites; Cell Differentiation; Humans; Leukemia, Promyelocytic, Acute; Methylation; Molecular Structure; Retinoids; Structure-Activity Relationship