Table 1 Solutions for resistance to decitabine and 5-azacytidine evaluated in vivo.

1.

↑Dose

– Overwhelm resistance mediated by DCK, UCK2, CDA and CAD expression changes

Cytotoxic to normal sensitive tissues (bone marrow) but failed to deplete DNMT1 in malignant tissues with ↓DCK, ↑CDA and ↑CAD [3, 26] (poor therapeutic index)

2.

Frequent, distributed schedules of administration

– ↑ Overlap between drug exposure windows and malignant S-phase entries

–- Avoid troughs in DCK/UCK2 expression induced by the pro-drugs

(a) Frequent, distributed administration of Dec 2–3X/week was superior to pulse-cycled administration for 5 consecutive days/month (Supplementary Fig. S6)

(b) Dec scheduled to avoid DCK troughs (Day 1,2 each week) was superior to scheduling that coincided with DCK troughs (Day 1,4 each week) (Supplementary Figs. S4, S5)

3.

Add tetrahydrouridine (THU)(CDA-inhibitor)

– Overcome CDA-imposed limits on Dec/5Aza plasma t_½ and tissue distribution

– Overcome CDA-imposed limits on Dec/5Aza intra-cellular t_½

– Counter auto-upregulation of CDA by Dec

(a) THU increased Dec or 5Aza systemic bioavailability approximately tenfold [40, 41] and increased intra-cellular t_½ [57,58,59]

(b) THU + Dec or THU + 5Aza (Dec/5Aza doses reduced by 50%) was superior to Dec or 5Aza alone (Figs. 4–6)

4.

Add hydroxyurea or thymidine (ribonucleotide reductase inhibitors)

– Counter competition from dCTP produced via de novo pyrimidine synthesis

Hydroxyurea or thymidine did not add benefit to Dec or THU/Dec (Fig. 4, Supplementary Fig. S6).

5.

Alternate Dec with 5Aza, timed to exploit reactive peaks in DCK and UCK2 expression

– Dec primes for 5Aza uptake and vice versa, effects that peak at 72–96 h

THU + Dec/THU + 5Aza alternated week to week (Fig. 6) was superior to THU + Dec or THU + 5Aza alone (Figs. 5, Supplementary Fig. S7), or THU + Dec/THU + 5Aza alternated month to month (Fig. 5) or given simultaneously (Supplementary Fig. S7)