MAPK Expression Profile in AML cells ex Vivo changes with Differentiation Induced by 1,25-Dihydroxyvitamin D3 and Its Analogs PRI-1906 and PRI-2191

The physiological form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), has well-established anti-cancer activity, but its potential clinical use is limited by hypercalcemia. Efforts to overcome this difficulty include structural modifications of 1,25D, as well as enhancement of its activity by other compounds such as antioxidants. The PRI analogs (termed “deltanoids”) have been shown to have low calcemic activity in animals, and while the PRI-1906 (24-ene-1,25-dihydroxyvitamin D2) and PRI-2191 ([24R]-1,24-dihydroxyvitamin D3) have greater activity on established Acute Myeloid Leukemia (AML) cell lines than their congeners, their relative potency on differentiation of AML blasts is not entirely clear. We therefore studied the effects of these analogs on leukemic blasts obtained from AML patients with different FAB subtypes, and combined them in some cases with Carnosic Acid (CA), a natural benzenediol abietane diterpene found in rosemary (Rosemarinus officinalis) and common sage (Salvia offinalis).

Leukemia blasts from patients with newly diagnosed, untreated AML were obtained from the peripheral blood after formal consent on an IRB approved protocol. Cells were isolated by Histopaque centrifugation, washed, and placed in tissue culture. 1,25D, PRI-1906, or PRI-2191 (each 10 nM), and CA (10 uM) were added, and cells cultured for the times shown, and then harvested for analysis.

Cells from 8 patients were studied (1 patient with FAB-M1, 1 patient with FAB-M2, 1 patient with FAB-M2 with mixed myeloid and lymphoid lineage; 1 patient with FAB-M3; 1 patient with FAB-M4; 3 patients with FAB-M5). Using expression of CD11b/CD14 by immunophenotyping as markers of differentiation, the 1,25D and the deltanoids each induced modest differentiation in AML FAB-M1, with greater differentiation in response to the combination of a deltanoid plus CA. Similar differentiation effects were seen in AML FAB-M2, and more dramatic effects in AML FAB-M3. Differentiation was also seen in AML FAB-M4. In AML FAB-M5, 1,25D and deltanoids induced differentiation, but CA appeared to add little to this process in 2 of the patient's samples. One patient with AML FAB-M5 did show significant differentiation in response to CA as well as 1,25D; of note, that patient had a t(11;19) involving the MLL-1 gene, with normal FLT-3 genotype). In 6 out of the 8 cases, the G1 arrest increased when AML cells were treated with CA alone, but the effect was not enhanced by CA combined with 1,25D or its analogs. MAP Kinase mRNA array analysis showed upregulation of MAP Kinase pathway molecules in response to 1,25D and the deltanoids.

The analogs of 1,25D, PRI-1906 and PRI-2191 induce cell cycle arrest and differentiation of AML cells ex vivo, and carnosic acid can enhance this effect. 2. As previously shown for 1,25D, MAPK signaling network is an important regulator of this process. 3.Since the PRI analogs are less calcemic than 1,25D, and carnosic acid has no effect on calcium homeostasis in AML cells, the potential therapeutic ratio is higher for the analogs combined with carnosic acid enhancer. Thus, these compounds may be considered as candidates for chemoprevention/ differentiation therapy of AML. (Supported by NIH Grant 1RO1-CA044722-21 to GPS).

No relevant conflicts of interest to declare.