Iron Overload, Heme Oxygenase-1 Expression and Drug Resistance in Acute Leukemia

Liis Lepik¹, Cristina Dumitrescu², Simon Steiner³

Introduction

populations it causes organ failure unless chelated [3,4].

described as a surrogate measure of iron overload [1,7,8].

Ferritin is not, however, an iron assay. It rises during inflammation and macrophage activation as well as with iron loading [9,10], and both processes are present in a patient with active leukaemia receiving cytotoxic therapy. Separating the two matters because chelation addresses one and not the other [2,11].

Heme oxygenase-1 connects these questions. The enzyme degrades heme into biliverdin, carbon monoxide and ferrous iron, so it is a source of intracellular iron; it is also a canonical oxidative stress response gene overexpressed in acute myeloid leukaemia and implicated in resistance to chemotherapy [12,13,14].

Material and Methods

Search strategy: PubMed and the archives of the major haematology and transplantation journals were searched for studies of iron status and heme oxygenase-1 in leukaemia. Search terms combined acute leukaemia and myelodysplastic syndrome with serum ferritin, iron overload, transfusion burden, liver iron concentration, labile plasma iron, chelation, heme oxygenase-1 and chemoresistance. Reference lists of retrieved cohorts and laboratory studies were screened by hand.

Eligibility: Studies were included if they reported the association between iron status or heme oxygenase-1 expression and clinical outcome or drug sensitivity in a defined acute leukaemia or myelodysplastic syndrome population. Laboratory studies in cell lines and murine models are cited where they establish mechanism and are identified as such.

Data extraction and handling: Design, cohort size, method of iron assessment, threshold applied and reported effect estimates were extracted as published. The method of iron assessment is stated alongside every estimate, because the central question of this review is whether ferritin measures what it is taken to measure. No pooling or re-analysis was performed and no patient-level data were obtained. Figures 1 to 4 and Tables 1 to 3 reproduce published values only. This is a narrative synthesis and was not registered as a systematic review.

Results

What a raised ferritin predicts: A prospective non-interventional study across twenty centres reported outcomes for 298 patients with acute leukaemia, lymphoma or myelodysplastic syndrome receiving a first matched sibling allograft, using a threshold of 1,500 micrograms per litre set at the cohort median [9]. Above that value, overall survival (HR 2.5, 95% CI 1.5 to 4.1, p = 0.0005) and progression-free survival (HR 2.4, 95% CI 1.6 to 3.8, p < 0.0001) were inferior [9].

Excess mortality arose from both directions. Relapse incidence was higher (HR 2.2, 95% CI 1.2 to 3.8, p = 0.007) and so was non-relapse mortality (HR 3.1, 95% CI 1.5 to 6.4, p = 0.002), the latter driven by significantly higher infection-related mortality (HR 3.9, 95% CI 1.6 to 9.7, p = 0.003) (Figure 1) [9].

Retrospective work agrees on direction and magnitude. Raised pretransplantation ferritin was strongly associated with lower overall and disease-free survival, an association restricted on subgroup analysis to patients with acute leukaemia or myelodysplastic syndrome, with an absolute difference of 37 per cent in five-year survival between highest and lowest quartiles in the myelodysplastic

The endpoint it does not predict: In the same prospective study, neither the incidence nor the severity of acute or chronic graft-versus-host disease was associated with ferritin levels (Figure 2) [9]. That negative result is informative rather than incidental.

Graft-versus-host disease is the alloimmune complication of transplantation, whereas infection reflects the depth and duration of immune incompetence [15,16]. A marker tracking infection but not alloreactivity is pointing at host defence rather than at donor immunity, which is consistent with either iron-mediated impairment of neutrophil and macrophage function or with underlying inflammation [11,17].

Systematic review of ferritin and outcome in acute myeloid leukaemia reports the same pattern, with infection prominent among causes of death in patients above the threshold and an association between pre-transplant levels and relapse susceptibility that was not consistent across all studies examined [18].

Ferritin measured at diagnosis rather than before transplantation carries prognostic weight as well, which argues that at least part of the signal precedes transfusional loading [23]. A patient has received few units at that point, so a raised value at presentation is more plausibly disease-related inflammation than accumulated iron [17,23].

Principal reported

Study n Method finding

Lower survival; 37% Armand et al. 2007 Cohort Serum ferritin quartile difference in [7] myelodysplasia Ferritin and Alessandrino et al. Ferritin independent; Cohort transfusion 2010 [8] transfusion count not burden Magnetic Labile plasma iron Armand et al. 2011 Prospective resonance rare; hepcidin [10] imaging appropriate Enzyme silencing Expression and Wei et al. 2015 [13] 64 restores silencing chemosensitivity Infection-related Penack et al. 2020 Prospective, 298 mortality hazard ratio [9] multicentre 3.9 Enzyme drives Zhang et al. 2022 Co-culture and Laboratory escape from natural [19] murine killer cells Table 1: Principal studies reviewed, with method and the finding as reported by the original investigators What direct measurement shows: A prospective study used hepatic and cardiac magnetic resonance imaging to estimate iron burden in consecutive adults with acute leukaemia or myelodysplastic syndrome scheduled for myeloablative transplantation, alongside labile plasma iron and hepcidin in plasma and urine [10]. Ferritin correlated strongly with estimated liver iron concentration at r = 0.75, and that correlation was mostly dependent on prior transfusion history (Figure 3) [10].

Two further findings complicate the simple reading. Labile plasma iron elevation, which is the fraction held responsible for tissue injury through catalysis of free radical formation, was rare [10]. And serum hepcidin was appropriately elevated in patients with hepatic iron overload, indicating that the regulatory response to iron loading was intact rather than deranged [10].

Regression calibration in that study supported the hypothesis that raised pre-transplant liver iron concentration is significantly associated with inferior post-transplant survival [10], so iron burden does carry prognostic weight independently. What the data do not establish is that the ferritin signal is wholly attributable to it [2,9,10].

The enzyme and chemoresistance: Heme oxygenase-1 has been implicated in acquired resistance in solid tumours, and its role in acute myeloid leukaemia was examined by comparing marrow or blood from newly diagnosed (n = 26), relapsed (n = 10) and remitted patients (n = 18) against healthy donors (n = 10), with silencing experiments in cell lines (Figure 4) [13].

A cytarabine and daunorubicin-resistant line expressed markedly higher enzyme messenger RNA and protein than its sensitive parent, and downregulating the gene significantly enhanced sensitivity to chemotherapy. Hypoxia-inducible factor 1-alpha and glucose transporter 1 fell, caspase-3 and caspase-8 rose, and BCL-2 decreased [13]. Overexpression of all three genes was associated with poor response in patients [13].

Independent work established that the enzyme underlies resistance of leukaemic cells to tumour necrosis factor-induced apoptosis [12], and simultaneous targeting of the enzyme and of hypoxia-inducible factor 1-alpha has been shown to sensitise cells to cytarabine [20]. Enzyme expression is regulated through nuclear factor erythroid 2-related factor 2 among other pathways [14,21].

Figure 4: Composition of the cohort in which enzyme expression was compared [13]

Measure What it reflects What it predicts

Iron stores and Survival, relapse, Serum ferritin inflammation together infection death [7,9] Transfusion Not independent of ferritin Iron delivered burden [8] Liver iron by Tissue iron directly Inferior survival [10] imaging Labile plasma The injurious fraction Rarely elevated [10] iron Hepcidin Regulatory response Appropriately raised [10] Table 2: Measures of iron status and what each captures Iron after transplantation, not only before it: Ferritin does not stabilise once conditioning is complete. Levels rise further in the months after transplantation, peaking between one and six months, and post-transplant iron overload carries its own prognostic weight [24,26]. Management of iron burden in this population has accordingly been addressed as a question extending beyond the pre-conditioning measurement [25,27].

Hepcidin behaviour after transplantation reflects erythropoietic activity as well as iron status [28], which complicates interpretation during engraftment precisely when transfusion requirement is highest. That temporal complexity is one reason a single pre-conditioning value is a convenient rather than a complete assessment [10,24].

Iron in cancer biology more broadly: The relationship between iron and malignancy is not confined to transfusional loading. Iron requirements of proliferating cells, and the pathways tumours use to acquire it, have been reviewed as a therapeutic target in their own right [29]. Ferroptosis, an iron-dependent non-apoptotic cell death, was characterised as a distinct mechanism [30] and is the process the enzyme modulates in both directions [22].

Oxidative stress links this to leukaemia genetics. Internal tandem duplication of FLT3 raises reactive oxygen species production with increased DNA damage and misrepair [31], and cell lines carrying that lesion show upregulated enzyme and nuclear factor erythroid 2-related factor 2 expression [22,31]. Whether iron overload compounds that burden in patients has not been established [29,32].

Organ consequences of iron loading are best characterised in the heart, where both deficiency and overload impair function [32]. In leukaemia, cardiac risk is dominated by anthracycline exposure, so an additional iron-mediated contribution would be difficult to isolate without direct imaging [10,32,33].

Current

Question Evidence quality answer

Does raised ferritin Yes, robustly Prospective multicentre [9] predict death?

Does it reflect iron Imaging correlation r = 0.75 Partly burden? [10] Is the injurious iron Rarely Direct measurement [10] fraction raised?

Does the enzyme Silencing experiments Yes in vitro confer resistance? [13,20] Does chelation Unknown No randomised trial [2] improve survival?

Ferritin is an excellent prognostic marker and a poor iron assay, and the literature has tended to treat those as one property. Above 1,500 micrograms per litre, survival, progression-free survival, relapse and non-relapse mortality all worsen, with hazard ratios between 2.2 and 3.1 [9]. That predictive performance is not in doubt.

What the data do not establish is that iron is the operative agent. Ferritin rises with inflammation and macrophage activation as well as with iron loading [9,17]; the mortality it predicts is infectious rather than the cardiac and hepatic failure that iron overload causes in chronically transfused populations [3,4,9]; and direct measurement found the injurious labile fraction rarely elevated with an intact hepcidin response [10].

Why the distinction decides the intervention

If raised ferritin marks iron overload, chelation should improve outcome, and the absolute survival difference of 37 per cent between ferritin quartiles in myelodysplasia would make that a large prize [7]. If it largely marks inflammation and disease activity, chelation would remove the marker without touching the process, and the resources would be better directed at infection prevention [2,9].

The evidence does not currently settle this. Whether iron chelation can safely and effectively reverse iron overload in patients continuing to receive transfusions, and whether that would translate into improved survival, has been identified as requiring prospective randomised testing rather than assumed [2]. Retrospective association cannot answer it because the confounding runs in the direction of the hypothesis.

Where the enzyme fits

Heme oxygenase-1 is the point at which the two halves of this literature meet, and it is dual in a way that resists simple interpretation. It liberates ferrous iron from heme, so its induction raises the intracellular labile pool; it is also induced by oxidative and inflammatory stress, so its expression marks the same processes that raise ferritin [12,14,21].

Its effect on cell death is correspondingly double-edged. Iron released by the enzyme can drive ferroptosis through ferritin degradation, while the same enzyme protects leukaemic cells from oxidative injury and from immune clearance, with the balance depending on the metabolic state of the cell and its microenvironment [14,19,22]. Overexpression has been shown to drive escape from natural killer cell cytotoxicity through suppression of an activating ligand [19].

Study limitations

The limitations belong to the underlying literature. Ferritin thresholds differ between studies, with 1,000 and 1,500 micrograms per litre both in use, and cut-offs set at cohort medians are not transferable [9,18,23]. The prospective imaging study enrolled a modest number of patients and excluded some who could not tolerate the scan, which may select against the sickest [10]. Most evidence on transplant outcome is retrospective and single-institution, and confirmatory prospective trials have been called for repeatedly [2,7]. Laboratory findings on the enzyme derive largely from cell lines, with patient sample numbers in the tens [13,20]. And ferritin is measured at a single pre-conditioning timepoint while both iron burden and inflammation change substantially through transplantation [18,24].

Two gaps deserve naming. No randomised trial has tested whether chelation before transplantation improves survival, so the therapeutic inference from a robust prognostic marker remains untested [2,10]. And no study reviewed here reports outcome stratified simultaneously by ferritin and by an independent inflammatory marker, which is the analysis that would separate the two contributions [9,17].

Conclusion

Serum ferritin above 1,500 micrograms per litre before allogeneic transplantation predicts inferior overall and progression-free survival at hazard ratios of 2.5 and 2.4, with excess mortality from both relapse and non-relapse causes, the latter driven by infection at a hazard ratio of 3.9. Graft-versus-host disease shows no association. Direct measurement complicates the iron interpretation: ferritin correlates with liver iron at r = 0.75 largely through transfusion history, yet labile plasma iron is rarely raised and the hepcidin response is intact. Heme oxygenase-1 links the two lines of evidence, releasing iron from heme while also acting as a stress-response gene whose silencing restores chemosensitivity and reduces hypoxia-inducible factor 1-alpha and BCL-2. Whether the ferritin signal is iron, inflammation or both determines whether chelation would help, and that question has not been tested. Ferritin measured at diagnosis, before meaningful transfusion has occurred, already carries prognostic weight, which points toward inflammation as a substantial part of the signal [23,34].

Acknowledgements

The authors thank the transplant registries, imaging cohorts and laboratories whose published data form the substance of this article.

Funding

No dedicated funding from public, commercial or charitable sources supported this review.

Conflict of Interest

The authors declare no competing interests.

Ethics Statement

This article reviews previously published studies only. No new patient data were collected and no participants were recruited, so ethical approval was not required.

References

1. Pullarkat, V. (2010) Iron overload in hematologic malignancies and outcome of allogeneic hematopoietic stem cell transplantation. Haematologica, 95(4): 529-531.
2. Steensma, DP. (2011) The relevance of iron overload and the appropriateness of iron chelation therapy for patients with myelodysplastic syndromes: a dialogue and debate. Curr Hematol Malig Rep, 6(2): 136-144.
3. Brittenham, GM. (2011) Iron-chelating therapy for transfusional iron overload. N Engl J Med, 364(2): 146-156.
4. Olivieri, NF., Brittenham, GM. (1997) Iron-chelating therapy and the treatment of thalassemia. Blood, 89(3): 739-761.
5. Wang, W., Knovich, MA., Coffman, LG., Torti, FM., Torti, SV. (2010) Serum ferritin: past, present and future. Biochim Biophys Acta, 1800(8): 760-769.
6. Koperdanova, M., Cullis, JO. (2015) Interpreting raised serum ferritin levels. BMJ, 351: h3692.
7. Armand, P., Kim, HT., Cutler, CS., Ho, VT., Koreth, J., Alyea, EP., et al. (2007) Prognostic impact of elevated pretransplantation serum ferritin in patients undergoing myeloablative stem cell transplantation. Blood, 109(10): 4586-4588.
8. Alessandrino, EP., Della Porta, MG., Bacigalupo, A., Malcovati, L., Angelucci, E., Van Lint, MT., et al. (2010) Prognostic impact of pre-transplantation transfusion history and secondary iron overload in patients with myelodysplastic syndrome undergoing allogeneic stem cell transplantation. Haematologica, 95(3): 476-484.
9. Penack, O., Peczynski, C., van der Werf, S., Finke, J., Ganser, A., Schoemans, H., et al. (2020) Association of serum ferritin levels before start of conditioning with mortality after alloSCT: a prospective, non-interventional study of the EBMT Transplant Complications Working Party. Front Immunol, 11: 586.
10. Armand, P., Sainvil, MM., Kim, HT., Rhodes, J., Cutler, C., Ho, VT., et al. (2011) Iron overload in patients with acute leukemia or MDS undergoing myeloablative stem cell transplantation. Biol Blood Marrow Transplant, 17(6): 852-860.
11. Ganz, T., Nemeth, E. (2015) Iron homeostasis in host defence and inflammation. Nat Rev Immunol, 15(8): 500-510.
12. Rushworth, SA., MacEwan, DJ. (2008) HO-1 underlies resistance of AML cells to TNF-induced apoptosis. Blood, 111(7): 3793-3801.
13. Wei, S., Wang, Y., Chai, Q., Fang, Q., Zhang, Y., Wang, J. (2015) Heme oxygenase-1 plays a crucial role in chemoresistance in acute myeloid leukemia. Hematology, 20(7): 384-391.
14. Chiang, SK., Chen, SE., Chang, LC. (2018) A dual role of heme oxygenase-1 in cancer cells. Int J Mol Sci, 20(1): 39.
15. Ferrara, JLM., Levine, JE., Reddy, P., Holler, E. (2009) Graft-versus-host disease. Lancet, 373(9674): 1550-1561.
16. Tomblyn, M., Chiller, T., Einsele, H., Gress, R., Sepkowitz, K., Storek, J., et al. (2009) Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol Blood Marrow Transplant, 15(10): 1143-1238.
17. Kernan, KF., Carcillo, JA. (2017) Hyperferritinemia and inflammation. Int Immunol, 29(9): 401-409.
18. Cheng, PP., Sun, ZZ., Jiang, F., Tang, YT., Jiao, XY. (2024) Impact of serum ferritin and iron overload on acute myeloid leukemia outcomes: a systematic review and meta-analysis. Ann Hematol, 103(11): 4407-4419.
19. Zhang, T., Fang, Q., Liu, P., Wang, P., Feng, C., Wang, J. (2022) Heme oxygenase 1 overexpression induces immune evasion of acute myeloid leukemia against natural killer cells by inhibiting CD48. J Transl Med, 20(1): 402.
20. Rahimi, A., Esmaeili, Y., Dana, N., Manian, M., Mirzaei, Z., Sharifi, M., et al. (2024) The sensitivity of acute myeloid leukemia cells to cytarabine is increased by suppressing the expression of heme oxygenase-1 and hypoxia-inducible factor 1-alpha. Cancer Cell Int, 24(1): 216.
21. Loboda, A., Damulewicz, M., Pyza, E., Jozkowicz, A., Dulak, J. (2016) Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell Mol Life Sci, 73(17): 3221-3247.
22. Yusenko, MV., Trentmann, A., Casolari, DA., Abdel Ghani, L., Lenz, M., Horn, M., et al. (2023) Susceptibility of acute myeloid leukemia cells to ferroptosis and evasion strategies. Front Mol Biosci, 10: 1275774.
23. Tachibana, T., Andou, T., Tanaka, M., Ito, S., Miyazaki, T., Ishii, Y., et al. (2018) Clinical significance of serum ferritin at diagnosis in patients with acute myeloid leukemia: a YACHT multicenter retrospective study. Clin Lymphoma Myeloma Leuk, 18(6): 415-421.
24. Meyer, SC., O’Meara, A., Buser, AS., Tichelli, A., Passweg, JR., Stern, M. (2013) Prognostic impact of posttransplantation iron overload after allogeneic stem cell transplantation. Biol Blood Marrow Transplant, 19(3): 440-444.
25. Sivgin, S., Eser, B. (2013) The management of iron overload in allogeneic hematopoietic stem cell transplant recipients: where do we stand? Ann Hematol, 92(5): 577-586.
26. Majhail, NS., Lazarus, HM., Burns, LJ. (2008) Iron overload in hematopoietic cell transplantation. Bone Marrow Transplant, 41(12): 997-1003.
27. Kanda, J., Mizumoto, C., Kawabata, H., Tsuchida, H., Tomosugi, N., Matsuo, K., et al. (2009) Serum hepcidin level and erythropoietic activity after hematopoietic stem cell transplantation. Haematologica, 94(12): 1697-1700.
28. Torti, SV., Torti, FM. (2013) Iron and cancer: more ore to be mined. Nat Rev Cancer, 13(5): 342-355.
29. Dixon, SJ., Lemberg, KM., Lamprecht, MR., Skouta, R., Zaitsev, EM., Gleason, CE., et al. (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell, 149(5): 1060-1072.
30. Sallmyr, A., Fan, J., Datta, K., Kim, KT., Grosu, D., Shapiro, P., et al. (2008) Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML. Blood, 111(6): 3173-3182.
31. Zhang, H., Zhabyeyev, P., Wang, S., Oudit, GY. (2019) Role of iron metabolism in heart failure: from iron deficiency to iron overload. Biochim Biophys Acta Mol Basis Dis, 1865(7): 1925-1937.
32. Döhner, H., Wei, AH., Appelbaum, FR., Craddock, C., DiNardo, CD., Dombret, H., et al. (2022) Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. Blood, 140(12): 1345-1377.
33. Kantarjian, H., Kadia, T., DiNardo, C., Daver, N., Borthakur, G., Jabbour, E., et al. (2021) Acute myeloid leukemia: current progress and future directions. Blood Cancer J, 11: 41.
34. Konopleva, MY., Jordan, CT. (2011) Leukemia stem cells and microenvironment: biology and therapeutic targeting. J Clin Oncol, 29(5): 591-599.