Pathogenesis of acute promyelocytic leukemia

Symposium organized by Hugues de Thé from 23 to 28 June, 2001.

Participants

Oliver Bischof (Institut Pasteur, Paris), Zhu Chen (Shanghai Second Medical University, Shangai, Chine), Christine Chomienne  (Hôpital Saint-Louis, Paris), Laurent Degos (Hôpital Saint-Louis, Paris), Ronald M. Evans (The Salk Institute for Biological ѕtudiеs, San Diego, USA), Paul Freemont (Imperial College of Science, tесhnology and Medicine, Londres, Grande-Bretagne), Scott Kogan (Comprehensive Cancer Center, San Francisco, USA), Michel Lanotte (Hôpital Saint-Louis, Paris), Jonathan Licht (Mount Sinaï School of Medicine, New York, USA), Wilson Miller (Hôpital général juif Sir Mortimer B. Davis, Montréal, Canada), Giuseppe Pelicci Pier (European Institute of Oncology, Milan, Italie), Ellen Solomon (Guy’ Hospital, London Bridge, Grande-Bretagne), Daniel Tenen (Harvard Institute of Medicine and Harvard Medical School, Boston, USA), Hugues De Thé (Hôpital Saint-Louis, Paris), Yi Wang Zeng (Shanghai Second Medical University, Shanghai, Chine), Arthur Zelent (Leukemia Research Fund Center, Londres, Grande‑Bretagne), Jun Zhu (Hôpital Saint-Louis, Paris)

Summary

The purpose of this meeting was to summarize the knowledge accu­mulated over ten years in the field of acute promyelocytic leukemia (APL) and to put into perspective this first model of differentiation — and oncogene-directed thеrapy. The participants have outlined a consensus of our current understanding of this malignancy and addressed the opened questions and ongoing work on the issues to be clarified. The wonderful setting of La Fondation des Treilles was instrumental in raising passion-ate discussion and new hypotheses in the understanding of this leukemia, from the laboratory bench to the hospital bed.

Report

Prof. Wang gave a review of the clinical treatment of APL with retinoic acid (RA). The first patients were treated in Rui Jin hospital in 1986 and were reported upon in 1987. RA induces the differentiation of the leukemic clone in vivo. This is accompanied by hyperleucocytosis in 60% of the cases and the Retinoic Acid Syndrome in 5%. Adding chemotherapy to RA-treatment consistently increases the complete remission rate which then reaches 80%. The leukemic cells secrete an unknown soluble inhibitor of normal haematopoiesis, which is rapidly reduced upon treatment. The tissue factor, involved in hypеrcoagulation, is very rapidly decreased upon RA treatment. Thus, consistent with the Confucius philosophy of equilibrium between Ying and Yang, RA cor­rects the defects associated with leukemia development and restores the normal bone marrow.

Prof. L. Degos addressed the clinical effects of RA on the most immediate danger of APL, coagulopathia. The latter results from the simulta­neous activation of thrombin and plasmin, resulting in fibrinogenopenia. Arsenic is faster than RA in correcting the leukemia-associated disorders. Resistance to retinoic acid as a single agent is constantly observed. This is often a relative resistance, because leukemic blasts remain sensitive ex vivo, in particular to 9 cis RA. In this respect, liposomal ATRA, which is not degraded by the liver at first passage, appears clinically much better and yields higher serum concentrations. In any case, a combined RA and chemotherapy induction regimen are essential and maintenance therapy is also important for long-term survival. Factors that point a negative outcome are initial tumor mass, the presence of a bcr3 breakpoint, and age, which favours early death.

Prof. Chomienne presented a set of analyses based on ex vivo differen­tiation of primary APL blasts.  She showed that a rapid ex vivo differenti­ation was predictive of a favorable long term outcome. The basis for this phenomenon is not understood, but it clearly points to the key role of dif­ferentiation in the eradication of the disease. She also showed that G-CSF secreted in response to RA at the onset of differentiation accelerated the maturation process. Other acute myeloid leukemias (AMLs), including those associated to t(8,21) translocations, are more primitive than APLs. Indeed, in APLs, normal stem cells can be separated from leukemic blasts by sorting. Antibodies to CD44 induce the differentiation of APL blasts, as well as caspase activation, which constitutes a new differentiation path­way. Vitamin D3 also differentiates 25% of the patient’s samples, raising the question of the use of associations between these two agents. FLT3 duplications also seems to be indicative of a poor outcome.

Prof. Chen presented a review on the effects of arsenic trioxide in APL. Arsenic was a cornerstone of the pharmacopea of Li Shi Zhen in 1518. It was prescribed against asthma and as a stimulant. In the 19’h century, arsenic was the main constituent of Fowler’s solution ini­tiallу developed for periodic fever. A group in Harbin used a mixture of herbs and arsenic for the treatment of skin cancer and leukemia with miracle results in APL (65% complete remissions, 25% cure at 10 yeаrs). Today, the 30% APL cases that relapse after combined RA/chemotherapy are eligible for arsenic, which is given by continuous perfusion. Newer trials have shown that there is no cross-resistance with RA, no marrow suppression, no seсondary cancers in 30 years and a significant effect on the APL-associated сoagulopathy, even better than that of RA. In vivo, arsenic triggers a partial differentiation and can cure some patients as a single agent. At the biological level, the differentiation marker CD11b is consistently (although modestly) induced and fresh APL blasts do not undergo apoptosis. In contrast, addition of other differentiation inducers such as cAMP induces terminal differentiation. This is accompanied by a massive decrease in E2F activity and increase in p21 expression. Finally, Dr. Chen showed that methylated arsenic induced apoptosis without degrading PML/RARa and was devoid of clinical effects.

Неlеn Solomon focused her presentation on two distinct issues: i) the target cеll that is immortalized by the translocation is not a primordial cеll, ii) the еpidemiology of the leukemia indicates a single rate-limiting step. Shе showed that APL patients without typiсal t( 15; 17) transloca­tions nevertheless express PML/RARa and acсordingly exhibit a microspeckeled PML pattern. The reciprocal fusion RARa/PML is found in 75% of the patients and trisomy 8 in 15% of them. Hence, the recip­rocal RAR/PML fusion is unlikely to be vert’ important in the pathogenesis of the disease. Intеrestingly, CD2, a T cell-marker can be found on APL blasts, particularly in the hypogranular variants. These represent a more primitive cеll, because upon RA exposure it differentiates with granules in a typical APL morphology.

D. Tenen addressed the possible involvements of CEBPa and e, as well as PU-1, in APL pathogenesis. These factors control the expression of key target genes such as the G-, GM- aid M-CSF receptors. CEBPa K.O. miсe have an acute myеloid leukemia (AIL)-like phenotype, while the b aid e genes are required for maturation to macrophages and granulocytes respeсtivеly. Moreover, CEBPa mutations have been found in AMLs. Cells from CEBPa K.O. mice can be induced to differentiate with IL3 or GM-CSF, pointing to the existence of parallel pathways of differеntiаtiоn. Interestingly, CEBP b is rapidly induced by RA in APL and could be one of the key target genes controlling differentiation. At least part of the sig­naling involves the Mусgene.

M. Lanotte presented a non-conventional view of APL in which the PML/RARa oncogene does not block differentiation, but rather induces the proliferation of undifferentiated cells. Secondary events then block the differentiation, possibly by rendering the cells resistant to the action of growth factors, yielding leukemia. One such pаthway is likely to be cAMP dependent because inhibitors of PKA will block RA-triggered dif­ferentiation and may induce apoptosis. Analyses derived from two mutant cell-lines R1 and R2 provide interesting perspective into the pathogenesis of the disease. R1 has an intact RA—signaling and yet fails to differentiate; in contrast, R2 that have a mutation in the RA-binding domain of PML/RARa fail to respond to both RA and сАМР, suggesting for a molecular link between these two signaling pathways. Interestingly, cAMP modulates the expression of the PML partner of the translocation. Finally, RXR agonists can also bypass the differentiation block, at least in the presence of CAMP and high serum concentrations.

Wilson Miller showed that the oncogenic PML/RARa can directly induce the TGIIase gene. PML/RARa bind the corepressor complexes much more avidly than the normal RXR/RAR heterodimer. Only very high RA concentrations will dissociate it. Similar to cAMP, TNF can strongly synergize with RA and even differentiate RA-resistant cell-liner. Among relevant target genes are p21 and HoxAl. As far as arsenic is con-cerned, Wilson Miller showed that the two agents have antagonistic effects ex vivo, although in the presence of cytokines or in vivo, arsenic is most likely an activator of differentiation.

Dr. Zelent showed that the shift from heterodimerisation to homo­dimerisation is a common theme in myeloid leukemias. Не then addressed the issue of modulation of RAR activity and expression in myeloid cells to show that RARa expression favours granulocytic differ­entiation and vice versa, which is presumably dependent on activation of the AF1 function of RARa. The RXR/PKA pathway also seems to be effective in clinical samples from t(11,17) patients. Finally, interference was described between the GАТA2 pathway aid PML or PLZE

Dr. J. Licht addressed that pathogenesis of t(11,17) associated APL. Не demonstrated that PLZF induces a cell-cyclearrest, but that the recip­rocal RARa-PLZF fusion accelerated cell-cycle progression through acti­vation of the cyclin A gene. The role of the reciprocal translocation prod­uct on the cell cycle suggests a model in which the two fusion proteins contribute to leukemogenesis by targeting distinct biochemical pathways. In this respect, the reciprocal product is always present in t(11,17) translocation, in contrast to the classical form of the disease (see above). 1с is also repressed by PLZE A comparison between the BTB domains of Вс1-6 and PLZF was also presented, together with the evidence that two distinct domains contribute to transcriptional repression in members of this gene family.

Dr. Kogan described in detail the onset of APL in transgenic mice. The disease starts to appear at 150 days and is ассоmрaniеd by a poorly understood skin phenotype. It starts with a modest differentiation block in the marrow, accompanied by a proliferation of promyelocytes. The promyelocytes then get progressively more autonomous, differentiate less and less and acquire the ability to invade other organs. Proliferation can be enhanced by addition of IL3 or activation of the FLT3 receptor. Retroviral transduction of CEBPa or e activates maturation.

Dr. Freemont focused on the normal function of PML. He discussed the function of the RING finger on which he performed extensive muta­genesis. This domain mау participate in the transfer of SUMO on neigh- boring proteins. Interestingly, many sumolation sites are immediately adjacent to NLS. Sumolation appears to modify the localisation of Daxx, which becomes cytoplasmic, a process observed in response to radiations. TUI proteins are also found in hexamers and a rough calculation indi- cates that over a million PML molecules are present in a body. At the cell biology level, these bodies are often adjacent to the МНС locus, which is itself adjacent to Dam. Hе also discussed the role of branching of SUMO-2 residues, for example with respect to protein degradation.

Dr. Bischof presented some work on the role of PML isoforms to induce senescence in primary human fibroblasts. Hеshowed, surpris­ingly, that despite the fact that all isoforms recruit p53, only PML IV induces senescence in a SUMO-indepеndent manner. He also showed that PML colocalizes with DNA repair protein such as Bloom and Rad5&, in particular alter irradiation.

Dr. Evans exposed the role of enhanced corepressor binding in APL. Hе then provided some evidence Chan the DNA-binding of PIL/RARa was considerably more relaxed, in terms of sequence specificity, than the normal RXR heterodimers. Finally, he presented an outline of the role of PPAR signaling with respect to macrophage differentiation. This was dis­cussed in view of the use of such agents in the treatment of diabetes and the possible interference with atherosclerosis.

Dr. Pellici exposed elegant experiments aimed at obtaining genetic information from in vivo pathogenesis. To do this, he developed retrovi­ral vectors that allow PML/RARa expression in the bone marrow of transplanted mice and yield to the development of leukemia. Hе also showed that PML/RARa expression induces resistance to a variety of stresses, such as irradiation or oxidative shock. The results strongly sug­gest that PML controls survival, while RAR controls differentiation. The relation between PML and P53 were detailed and PML shown to control Р53 acetylation and hence function. Finally, evidence was presented to show that NPM, another translocation partner, is a chaperon for P53 which binds both P53 and ARE

Drs. Zhu and de Thé analyzed in detail the requirements for the catab­olism of the PML/RARa oncogene and showed that the sumolation of PML is essential for its arsenic-induced degradation, but not for its trans­fer to the nuclear matrix. The links between the activation of nuclear receptors and their degradation was also extensively discussed in view of novel data that mechanistically link the two events. The role of PML/RARa degradation in the remission process was discussed. Data from animal models that show a clear synergy between the two agents and the similar clinical course of the two treatment, both strongly suggest that degradation of the oncogene plays a role in disease remission.

General discussions then addressed the major clinical and biological issues in this model malignancy. 10w can the imminency of death be controlled? How can we stimulate the immune response against the leukemia to increase long term survival? What are the available therapeu­tic agents? Retinoic acid (oral or probably better, liposomal), arsenic and chemotherapy. What is the optimal sequence in which these agents should be given? The fast that in the Chinese trials, two patients died of liver failure upon arsenic thеrapy has blocked the use of front-line arsenic, which would be a logical choice. Perhaps it should be used in elderly patients or those presenting hyperleucocytosis. Is it time for dose desescaladation? Among newer agents, histone desacеtylase was exten­sively discussed, but activators of cAMP transduction were also consi­dered, as well as newer retinoids. As for the basic biology questions, those deemed essential dealt with the nature of the critical RA-targets in APL, the exact mechanism of the РML/Р53 interaction, the real use of HDAC inhibitors, extension of the concepts drawn from the study of APL to other myeloid leukemias.