Topoisomerase IIα (topo IIα) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIα, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIα gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIα promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIα promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIα gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIα mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIα by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIα promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIα in tumors.

Background. It seems that the population of leukemia stem cells (LSCs) have fundamental importance in the origin and maintenance of the acute myeloid leukaemia (AML). Eradication of LSCs is a new goal of AML therapy. We hypothesized, that by monitoring of minimal residual disease (MRD) and its dynamics in different compartments (peripheral blood, PB; bone marrow, BM; sorted CD34+ BM cells; and CD34- BM cells) it would be possible to find some patterns reliably predicting clinical relapse. Aims. To find which compartment is the best for MRD monitoring and whether it would be possible to treat the disease in the phase of molecular relapse in order to prevent the hematological relapse. Methods. MRD monitoring, in average once or twice per month, was performed in all phases of treatment, and was done even more frequently in the cases of unstable MRD. RQ PCR for fusion transcripts (CBFB/MYH11, RUNX1(AML1)/ETO or fusion transcripts of MLL gene) and WT1 gene was used. Molecular relapse was defined as reappearance of the fusion transcript detection or its 10-fold increase, repeatedly detected. Some patients with already known MRD dynamics and high probability of imminent hematological relapse were treated at the time of molecular relapse. Results. In 67 AML patients and 6 healthy volunteers, 2352 BM or PB samples were examined, including 265 samples from CD34+ BM cells. Follow up was 31–287 weeks (median: 113 w). The correlation between the fusion transcripts levels in BM and PB was excellent (r=0,9676). The correlation between WT1 PB and BM levels was far less satisfactory. Since the WT1values did not mostly reach zero values even if the level of fusion transcript was 0, we wanted to find some “normal” value for WT1. Using the ROC curves, however, we were not able to find any WT1 level being a confidential marker of molecular remission in either compartment (PB, BM, CD34− or CD34+). In relapsed patients, the time from molecular to haematological relapse was 8 – 79 days (median: 25 d). In the cases of subsequent development of haematological relapses, the levels of fusion transcript in CD34+ BM cells were one order of magnitude higher than in the BM or PB, even in the case of CD34- blasts. Nine patients were treated for 17 molecular relapses with following results: chemotherapy, CR=2, PR=3, NR=1; gemtuzumab ozogamicin, CR=3, PR=1, NR=3; IL-2±DLI, CR=3, NR=1 (PR was defined as a decrease in fusion transcript level at least 10-fold, CR as a decrease to 0). Patients with CD33- blasts at diagnosis did not respond to gentuzumab ozogamicin. Non-responsiveness to one treatment option did not mean non-responsiveness to another treatment. Conclusion: Frequent quantitative monitoring (especially in CD34+ BM cells) of fusion transcripts (in contrast to WT1) is useful for reliable prediction of haematological relapse in AML patients. PB seems to be sufficient for frequent outpatient MRD monitoring. Efficient targeting of LSCs will be essential for AML cure, however, the best method is currently not known. Some now available procedures are sometimes surprisingly successful.

Patients and Methods. In order to find some markers, which reliably enable to predict clinical relapse in AML patients, we primarily focused on patients with the known fusion transcript (CBFB-MYH, AML1-ETO, or involving the MLL gene) and correlated this values with the WT1 for estimation of the true value of WT1 monitoring of disease behavior in a given patients. The value of different compartments for minimal residual disease (MDR) monitoring (PB, BM or CD34+ BM cells) was also analyzed. The study was prospective; in the case of MDR dynamics, the patients were actively called for earlier visit. This strategy could estimate the real time interval from MRD level increase to hematological relapse detection. In the interventional part of the study, the patients with already known MRD dynamics were treated at the time of molecular relapse. Results and Discussion. In 67 AML patients and 3 healthy volunteers, 2184 BM or PB samples were examined, including 240 samples from CD34+ BM cells. Follow-up was 31–252 weeks (median: 88 w). The correlation between the fusion transcripts levels in BM and PB was excellent (r=0.9676). The correlation between WT1 PB and BM levels was far less satisfactory. Since the WT1 values were frequently >0 even if the level of fusion transcript =0, we wanted to find some “normal” value for WT1. Using the ROC curves, however, we were not able to find any WT1 level being a confidential marker of molecular remission in either compartment (PB, BM or CD34+). Molecular relapse was defined as a reappearance of the fusion transcript detection or its 10-fold increase, repeatedly detected. The time from molecular to hematological relapse was 8–79 days (median: 25 d). In the cases of subsequent development of hematological relapses, the levels of fusion transcript in CD34+ BM cells were one order of magnitude higher than in the BM or PB, even in the case of CD34− blasts. Eight patients were treated for 13 molecular relapses with following results: chemotherapy, CR=2, PR=2; gemtuzumab ozogamicin, CR=3, PR=1, NR=3; IL-2±DLI, CR=2 (PR was defined as a decrease in fusion transcript level at least 10-fold). Patients with CD33− blast at diagnosis did not respond to gemtuzumab ozogamicin. Non-responsiveness to one treatment option did not mean non-responsiveness to another treatment. Conclusion: Fusion transcript monitoring enables reliable detection of molecular relapse in AML and high values in CD34+ BM cells signalize imminent hematological relapse (even in the case of CD34− blasts). PB is a suitable compartment for frequent monitoring. However, in some cases, relapse are fulminate, hardly allowing any intervention. WT1 does not seem to be a reliable marker for exact molecular relapse detection. AML at the stage of molecular relapse behaves similarly to AML at the frank hematological relapse: there are CRs, PRs, or NRs when using chemotherapy or gemtuzumab ozogamicin. AML with CD33− blasts at diagnosis does not seem to respond to gemtuzumab ozogamicin at the stage of molecular relapse. Success of AML therapy in the future seems to be dependant on efficient targeting the leukemia stem cell.