suppl.Table 1Population characteristics of the patients with acute myeloid leukemia

The patients were grouped according to the morphological subclass assignments (French-American-British classification) in columns versus the respective population parameters in rows. All 109 patients were measured at the time of diagnosis without prior treatment. 22 patients had undergone allogeneic stem cell transplantation (SCT recipients). The prognostic implication of the karyotype analysis at initial diagnosis was classified according to Grimwade (Grimwade et al. 1998).

suppl. Figure 1ABCA3 expression levels and treatment outcome in patients with AML with inclusion of stem cell transplant recipients, and cox regression analysis of ABCA3 expression levels and prognostic implication of the karyotype

Kaplan-Meier product-limit estimates of progression free survival (PFS, C), overall survival (OS, D) and OS separated for cytogenetic subgroups (A) for patients (n=109) with newly diagnosed acute myeloid leukemia treated with pediatric BMF and adult HD98 protocols, including stem cell transplant recipients. Cox regression analysis confirmed the strong prognostic implication of the karyotype findings. Accounting for the karyotype as a confounding variable, however, still did not alter the reduced hazard ratio (HR) for shortened overall and progressionfree survival for patients with low ABCA3 levels in this cohort(B). CI, confidence interval.

suppl. Figure 2Effects of ABCA3 on clonogenicity under chemotherapy ams association of drug resistance and ABCA3 expression in HL60 leukemia cells

Correspondingly to the MTT results, clonal regrowth on day 10 after exposure to chemotherapy with ABCA3 (A, survey left lower well and 100 x magnification right bottom panel) and without ABCA3 (A, survey left upper well and magnification 100x right upper panels) was significantly different for daunorubicin, etoposide, mitoxantrone and vincristine (A, right panel), but not for GO. ABCA3 did not protect HEK293 cells from the toxic effects of gammaradiation (B, left panels) or UVCradiation (B, right panels), both for direct toxicity and clonogenicity.

In HL60 leukemia cells with low endogenous ABCA3 expression, elevating ABCA3 expression by transient overexpression (C) increased the cellular resistance to daunorubicin, AraC and etoposide, compared with control cells transfected with a mutated, non-functional ABCA3-mutant N568K/GFP construct. CD33 positive HL60 were also efficiently protected from saturating concentrations of gemtuzumab-ozogamicin (GO) by ABCA3 (E). Vice versa, the reduction of endogenous ABCA3 by specific siRNA (D) sensitized HL60 cells to cytostatic damage by daunorubicin, Ara-C, and etoposide.

suppl. Figure3Detection of ABCA3 expression in hematopoietic stem cells

Immunocytology (A) and qRT-PCR analysis (B) revealed high levels of ABCA3 expression in early hematopoietic progenitor and stem cells. HEK293 (a-b) cells transiently overexpressing ABCA3 were used as positive, and wt HEK293 (c-d) as negative controls. ABCA3 expression was only detected in sorted CD34+ cells and SP cells, in contrast with CD34-and the majority of non-SP cells. Cell stains are represented at 100 x magnification in the left column of panels, and at 400 x magnification in the right column.