Induced differentiation of cancer cells: second generation potent hybrid polar compounds target cell cycle regulators

Hybrid polar compounds are potent inducers of differentiation of a wide variety of cancer transformed cells. Hexamethylene bisacetamide (HMBA) has been used as a prototype of these compounds to investigate their mechanism of action. Employing murine erythroleukemia (MEL) cells as a model, three characteristics of inducer-mediated commitment to terminal differentiation were demonstrated: (I) induced commitment was stochastic, requiring up to 5 cell cycles to recruit essentially all cells to commit to growth arrest in G1; (II) inducers caused a prolongation of the initial G1; and (III) the hybrid polar compounds induced a wide variety of transformed cells to terminal differentiation. These findings suggested that the rate limiting factor or factors for induction by these agents may be at the level of protein(s) regulating G1-to-S progression, which are common to most eukaryotic cells. It was found that HMBA induced a profound suppression of cyclin dependent kinase, cdk4, which reflected a marked decrease in stability of the protein, and is a critical change in the pathway of induced differentiation. HMBA also induced an increase in pRB and in the active, underphosphorylated form of this protein, an increase in the pRB related protein, p107, and an increase in the cyclin dependent kinase inhibitor, p21. Further, the free form of the transcription factor, E2F, was markedly decreased within hours of exposure of transformed cells to HMBA and found to complex with p107 and cdk 2. A phase II clinical trial was conducted using HMBA to treat patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. Of 28 patients, 9 patients achieved a complete or partial remission lasting from 1 to 16 months. These clinical studies also provided direct evidence that HMBA induces differentiation of transformed cells in patients. In four separate courses of treatment with HMBA, a patient with MDS and the monosomy 7 karyotype marking the malignant clone of bone marrow blast cells, achieved peripheral blood granulocyte counts that approached normal levels. More than 80% of these morphologically mature granulocytes carried the chromosomal marker characteristic of the malignant clone, providing clear evidence that the clinical response was consequent to maturation within the malignant clone. These phase II studies, however, also showed that HMBA is not a satisfactory therapeutic agent, owing to thrombocytopenia, which limits the amount of drug that can be administered. Furthermore, because continuous exposure to the agent is required for induction, and the biological half-life of HMBA in the patient is very short (about 1.5 hr), HMBA must be administered by continuous infusion to maintain a clinical effect. These factors, taken together, have inspired a continued search for additional hybrid polar compounds effective at significantly lower concentration and that might avoid the clinical side effects that limit the use of HMBA. We recently identified a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2M and 4M, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p21 protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of gemoglobin. Based upon their effective concentration, the cross-resistance or sensitivity of a HMBA-resistant MEL cell variant and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in planning future clinical studies toward the treatment of cancer by differentiation therapy