**9. The HIF pathway in leukemia**

again to ascorbic acid, with consumption of GSH; this may not be the case in acute myeloid

**Table 1.** The number of vital cells after 2 h of exposure to increasing concentrations of Vitamin C in the culture medium.

The cell lines used in this experiment are variants of human myeloid leukemia cells, and include: HL60, NB4, K562, U937, NB4-R1, NB4/As. It is evident that the total number of cells in culture decreases by increasing the concentration of Vitamin C in the culture medium. C = control (untreated) sample; VC = Vitamin C; VC 0.5 mM, VC 1 mM, VC 3 mM, VC

**HL60 (2 h) NB4 (2 h) K562 (2 h) U937 (2 h) NB4-R1 (2 h) NB4/As (2 h)**

Exp. 1 Contr. 471 912 663 1189 337 819

Exp. 2 Contr. 869 1020 694 829 936 958

Exp. 3 Contr. 843 1130 1020 969 967 859

VC 0.5 296 680 669 479 82.8 42.7 VC 1 108 494 628 245 39.7 31.9 VC 3 22.6 163 226 56.4 47 32.2 VC 5 15.1 143 82 30.2 35 48.4 VC 7 6.15 85.4 32.2 10.6 24.6 17.6

VC 0.5 423 959 399 704 624 823 VC 1 349 887 445 585 560 674 VC 3 217 337 200 232 335 581 VC 5 143 74.6 111 118 344 402 VC 7 89.5 147 35.2 93.7 255 329

VC 0.5 545 924 660 716 678 722 VC 1 438 835 507 689 668 649 VC 3 343 395 113 480 551 499 VC 5 218 346 35.2 411 443 453 VC 7 181 241 17.6 320 380 414

More importantly, the parallel exposure of normal hematopoietic precursors (CD34+), isolated from cord blood, to Vitamin C, at the concentrations that are cytotoxic for leukemic cells did not affect their survival, or impair their capacity to proliferate and differentiate in response to myeloid growth factors. These data confirm that Vitamin C is harmless for normal hematopoietic precursors and therefore highly selective in its anticancer/antileukemic effect.

Hypoxia and induction of hypoxia-inducible factors (HIF) is a hallmark of many tumors [78, 79].

**8. Hypoxia inducible factor (HIF): the forgotten pathway**

5 mM = Vitamin C at 0.5, 1, 3, and 5 mM in the culture medium.

leukemia.

164 Myeloid Leukemia

The role of HIF-1α in leukemia, and in particular in acute myeloid leukemia (AML), has only recently emerged and it is still somewhat controversial. One possible explanation for this delayed interest in the role of hypoxia in leukemia could be the fact that leukemia is not considered a "solid" tumor, and therefore, the role of oxygen, in its pathogenesis, has been considered inconsequential for long time. This erroneous view, has been recently reviewed, as data have emerged, demonstrating that leukemic cells are sensitive to the oxygen tension, and hypoxia influences leukemic cell proliferation, differentiation, and resistance to chemotherapy [98].

H2 O2

**11. What to do next?**

both in vitro and in vivo.

 production. Asc 2-P added to the cells in culture is stable and releases Vitamin C by plasma membrane alkaline phosphatase hydrolysis [120]. Therefore, Asc 2-P allows a better character-

toxic effects of the native molecule. Asc 2-P treatment of the IDH1 AML-mutant mice induced an increase of 5hmC levels, a reduction of leukemic proliferation and an increase in expression of genes involved in leukocyte differentiation [118]. The stimulatory effect of Vitamin C on myeloid differentiation is mediated though the restoration of a normal expression and function of transcription factors, such as PU.1 and RUNX1, required for normal myeloid differentiation. A second study provided clear evidence that in various leukemia models, Vitamin C treatment induces the restoration of TET2 function, blocking aberrant self-renewal and leukemia progression. Treatment with Vitamin C, mimics TET2 restoration, driving DNA hypomethylation and, by enhancing 5hmC formation, suppresses leukemic colony formation and leukemic progression of primary human leukemia patient-derived xenografts (PDXs). Interestingly, TET2-mediated DNA oxidation induced by Vitamin C-treated leukemic cells, greatly enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to target TET-deficient leukemic cells. These observations suggest that future clinical trials could incorporate high-dose Vitamin C as an adjuvant to standard che-

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167

ization of the epigenetic activity of Vitamin C, without the "disturbing" H<sup>2</sup>

motherapy/demethylating therapy, particularly in TET2-deficient neoplasms [121].

The anticancer properties of Vitamin C are known, since at least six decades, even though its use in clinical practice has only recently re-emerged, after the demonstration that in relatively high concentrations, it can selectively kill a number of different human tumor cells,

The proof of the anticancer efficacy of Vitamin C in high doses, administered by mouth, has been reported four decades ago, by Linus Pauling [54–57], and further confirmed, very

Vitamin C is a natural compound, and it is an antioxidant and a life-saving nutrient with multiple beneficial effects on the human body. Man, some primates, and a few other mammals do no longer produce it. Beyond being a natural and essential nutrient, Vitamin C shows, in vitro, an outstanding efficacy in killing a number of different cancer cells, with an efficiency

Vitamin C is extremely selective since it kills only cancer cells, by sparing, at the same time, all the other cells of the organism. As a consequence, it is very well tolerated, and devoid of any significant side effects. In fact, the only (relative) contraindication to its use, is the lack of the enzyme glucose-6-phosphate-dehydrogenase (G6PDH), a rare genetic condition also known as "favism." More importantly, within an expensive and often artificially inflated market, such as that of the anticancer drugs [122, 123], Vitamin C, with its low cost, represents an

that no other anticancer drug, presently available on the market, has ever shown.

outstanding opportunity for both the patients and the healthcare system.

recently, by experimental in vitro and in vivo data [30–32, 66, 69].

Migliavacca and Coll. have recently demonstrated oncogenic function of HIF-1α, in the M5 Fab subtype of AML [99]. In particular, the authors have demonstrated that in M5 AML, HIF-1α mediates the ability of leukemic cells to migrate and invade extramedullary sites. The same group has demonstrated that PML-RARα and other fusion proteins involved in the pathogenesis of acute promyelocytic leukemia (APL) behave as transcriptional coactivators of HIFs, and both HIFs and PML-RARα enhances the progression of APL, by promoting cell migration, homing to bone marrow, and bone marrow neo-angiogenesis [100, 101].

Further investigations [102] have demonstrated that HIF-1α plays critical and pleiotropic roles in the pathogenesis of chronic lymphocytic leukemia (CLL).

Globally, elevated levels of HIF-1α have been reported in AML [103–106], APL [100], acute lymphoblastic leukemia (ALL) [107], and chronic myelogenous leukemia (CML) [108, 109]. Furthermore, HIF-1α overexpression conditions disease severity and outcome in both AML and myelodysplastic syndrome (MDS) [110–112].

Overall, the available data show that hypoxia and HIF-mediated signaling play a crucial role in leukemia, and targeting HIF with specific drugs or natural inhibitors, such as Vitamin C, represents a potentially useful approach to its treatment [113].
