the different forms of acute myeloid leukemias ( amls ) * are characterized by the arrest of cell differentiation , the induction of cell proliferation , and the repression of normal hematopoiesis .
these malignancies are often associated with recurrent chromosomal translocations , most of which encode fusion proteins derived from transcription factors ( 1 ) .
functional analyses of several of these fusion proteins have shown that they behave like potent transcriptional repressors ( 2 ) .
repression is often achieved through the modification of chromatin structure by histone desacetylases which blocks the expression of unidentified genes that control myeloid differentiation .
the inhibition of desacetylases by a variety of compounds has been fairly effective in cell culture ( 3 ) and in animal models of leukemia ( 4 ) , but as yet , there only is slight evidence of its beneficial effects in clinical settings ( 5 ) . to date , the only real clinical success of transcription / differentiation therapy has been in acute promyelocytic leukemia ( apl ) , for which two drugs , retinoic acid ( ra ) and arsenic trioxide ( as2o3 ) induce remissions ( 6 , 7 ) . the action of these drugs is remarkable because they both target the oncogenic promyelocytic leukemia ( pml)retinoic acid receptor  ( rara ) fusion protein and reverse pml / rara  mediated repression ( 8) .
cyclic adenosine monophosphate ( camp ) also differentiates many aml cell line and strongly synergizes with other differentiating agents ( references 9 and 10 ; for a review , see reference 11 ) .
moreover , in apl cells , camp can decrease the concentrations of ra required for differentiation to almost physiological ones ( 12 ) .
however , a number of acute toxic reactions have precluded or severely limited trials using camp derivatives ( 13 ) . here , we used an animal model of apl derived from pml / rara transgenic mice ( 14 ) to study the effects of camp in vivo .
we showed that camp induces major cell growth arrest together with differentiation and synergizes with both as2o3 and ra to clear ra - sensitive or ra - resistant apl . in an apl patient who had become resistant to ra and as2o3 therapy , addition of theophylline ,
an inhibitor of camp intracellular degradation , induced a clinical remission . that activation of camp signaling is beneficial in apl , independently of its sensitivity to ra , raises hopes for the successful treatment of therapy - resistant forms of aml .
morphology and differentiation of the apl cell line nb4 were evaluated on may - grnwald giemsa 
8-chloro - adenosine 3 - 5 cyclic monophosphate ( 8-cl - camp ) and 8-(4-chlorophenylthio)adenosine 3 - 5 cyclic monophosphate ( 8-cpt - camp ; reference 15 ) were respectively obtained from the biology life research institute and sigma - aldrich .
8-cpt - camp was used at a concentration of 2.10 m. spleen - derived leukemic blasts ( 10 ) were serially passaged in syngeneic fvb / n mice 6 wk old , weighing 20 g , as described previously ( 14 ) . both ra - sensitive leukemias ( strain 935 ) or ra - resistant leukemias ( strain 4048 ; reference 16 ) were used .
all experiments involving mice were repeated between two and eight times , usually with two mice in each treatment arm .
0.5 l / h alzet pumps were loaded with 20 mg / ml 8-cl - camp and implanted subcutaneously on the back of treated mice .
aminophylline , a stabilized precursor of theophylline , was injected intraperitoneally ( 100 l / day of a 25 mg / ml solution ) .
ra and as2o3 treatments , autopsies , and cell or tissue analyses were performed as described previously ( 14 ) . for western blot analysis ,
plasma 8-cl - camp was measured by hplc using a c18 column ( chromosep inertil 5 ods3 ) with a 15% methanol/50 mm phosphate buffer , ph 5.85 , as a mobile phase and uv detection at 254 nm .
the patient studied gave informed consent to the use of theophylline to enhance ra / as2o3 differentiation .
the patient 's daily treatment consisted of 45 mg / m ra per os , 10 mg as2o3 intravenously , and 250 mg theophylline per os .
it is well known that camp greatly enhances the ra - induced differentiation of many cell lines derived from embryonal carcinoma or myeloid leukemias , including apl ( 10 ) . as low concentrations of as2o3
were found to induce only incomplete differentiation in an apl cell line ( 7 ) , we tested the hypothesis that camp would also enhance as2o3-induced differentiation .
even very low doses of as2o3 combined with 8-cpt - camp ( a low toxicity camp analog ) induced nbt reduction in 40% of the nb4 cells , whereas camp or as2o3 alone did not have significant effects ( unpublished data , see fig .
similarly , only combined as2o3 and 8-cpt - camp induced morphologic differentiation of nb4 cells into myelocyte - like cells ( unpublished data ) , as reported recently ( 17 ) . to assess the possible in vivo efficiency of camp , we used transplantation apl model ( 14 ) derived from ra - sensitive pml / rara transgenic mice ( 18 ) .
we developed a similar transplantation model for ra - resistant apl , using leukemic cells from pml / rara transgenic mice in which a point mutation in the transgene impairs the binding of ra to pml / rara ( 16 ) .
in vivo growth of this leukemia is much slower than in the ra - sensitive model , liver , or spleen invasion is not as pronounced and either ra or as2o3 have mild antiproliferative effects in the absence of significant differentiation ( see fig .
mice bearing established leukemias were treated with 8-cl - camp , as2o3 , ra , or combinations of these drugs and killed 17 d after treatment .
continuous 8-cl - camp infusions allowed significant plasma concentrations to be reached ( 1 m on an average at day 3 ) . despite its toxicity , 8-cl - camp induced major antileukemic effects in both ra - sensitive and ra - resistant apls , as assessed by spleen weight ( fig .
myeloid cells undergoing differentiation were always observed in the marrow of ra - resistant apl after 7 d of treatment , and were often found upon treatment of ra - sensitive apl ( fig .
1 ) . in leukemic cells infiltrating the liver , a sharp reduction in the number of mitoses was observed after 8-cl - camp treatment ( 3% post- versus 28% pretreatment ) and a few condensed nuclei were seen ( unpublished data ) .
these results show that in these ra - sensitive or ra - resistant mouse models of apl , camp triggers a combination of cell growth arrest , differentiation , and apoptosis , leading to dramatic regressions of the leukemia .
8-cl - camp induces growth arrest in ra - sensitive ( ra , a ) or ra - resistant ( ra , b ) apl in mice .
( top ) spleen weight after 7 d of 8-cl - camp treatment or no therapy (  ) .
( bottom ) liver from the same mice . since camp greatly increased as2o3-triggered differentiation ex vivo in the nb4 cell line ( fig . 2    a )
, we combined 8-cl - camp and as2o3 treatments in vivo . with this combination ,
the spleen , liver , and bone marrow of mice with ra - sensitive apl became free of leukemia between days 1 and 3 , whereas mice treated with as2o3 or camp alone retained a significant tumor burden consisting of differentiating leukemic cells ( fig .
2 , b , e , and f ) . note that erythroblasts and megakaryocytes were extremely numerous in a cell - rich marrow ( fig .
2 e ) , in agreement with the idea that camp promotes the regrowth of these cells . in keeping with our ex vivo results , camp synergized with as2o3 to trigger differentiation , as shown by cd11b expression on bone marrow blasts at day 1 ( fig .
2 c ) , strongly suggesting that enhanced differentiation of the leukemic blasts contributes to these accelerated remissions .
the level of the cdk inhibitor p21 , a known camp target implicated in growth arrest , differentiation , and apoptosis , rose strikingly in bone marrow apl blasts upon treatment in vivo ( fig .
synergy was also noted between 8-cl - camp and ra with respect to both spleen weight and marrow cell differentiation , although this synergy was dimmed by the stronger differentiating effect of ra ( fig .
3 )   . arsenic and camp synergize to induce tumor regression and differentiation in ra - sensitive apl .
( a ) 8-cpt - camp potentiates as2o3-induced nbt reduction at 4 d in the apl cell line nb4 .
( b ) in a mouse model of apl , continuous infusion of 8-cl - camp for 3 d greatly reduces spleen weight and synergizes with as2o3 ( as ) to induce tumor reduction ( one representative experiment ) .
( c ) cd11b expression on bone marrow cells after 24 h of in vivo treatments , as indicated .
( d ) 8-cl - camp induces p21 expression in leukemic bone marrow after 24 h of in vivo treatment . * cross - reactive protein .
3 d of combined treatment restores normal hematopoiesis in the bone marrow ( e ) and induces tumor clearance from the liver ( f ) .
( g ) theophylline ( t ) therapy ( 3 d ) induces blast differentiation .
( a ) spleen weight after 7 d of treatment or untreated (  ) .
8-cl - camp induced even more dramatic regressions in ra - resistant apl , with some morphologically complete clearances ( figs . 1 and 4 )   .
unexpectedly , major enhancements of leukemia clearance and differentiation were always observed when ra was combined with 8-cl - camp ( fig .
although as2o3 alone triggered a slight antiproliferative effect in the absence of significant differentiation , the ra / as2o3 combination triggered minor , but reproducible , differentiation ( fig . 4 b ) .
the stable camp derivative used here induces massive diuresis , precluding any long - term use , and may be further metabolized into potentially cytotoxic nucleotide analogs ( 15 ) . to ensure that the antileukemic effect indeed resulted from the activation of camp signaling , we used theophylline , a phosphodiesterase inhibitor which stabilized pools of endogenous intracellular camp . like 8-cl - camp ,
the - ophylline blocked growth in ra - sensitive apl and induced some apoptosis , accompanied by non - terminal differentiation ( fig .
as expected , the enhancement of differentiation was more pronounced with as2o3 than with ra ( fig .
however , there was no obvious boost in normal hematopoiesis , possibly reflecting low production of endogenous camp in normal cells .
on the basis of these promising results in mouse apl , an ra / as2o3-resistant apl patient was offered an experimental course of combined ra / as2o3/theophylline therapy .
previously , a month of the ra / as2o3 combination had led to a slow decrease in bone marrow blasts , a peak of differentiating myeloid cells in the blood ( fig .
5 )   , but normal hematopoiesis was not restored and the leukemia had reappeared 5 wk later ( fig . 5 , a and b ) .
however , with combined ra / as2o3/theophylline treatment , leukemic cells underwent differentiation and normal erythroblasts appeared rapidly ( fig . 5 , a and c ) .
the patient no longer required red cell or platelet transfusions and white blood cells and hemoglobin reached levels close to the normal ones .
the patient remained leukemia free for 4 mo , but then the leukemic clone reappeared .
paradoxically , with ongoing ra / as2o3/theophylline therapy and despite the leukemia relapse , normal hematopoiesis has been maintained to date ( 5 mo since relapse ) , an extremely unusual situation in apl , in which cytopenia is the first sign of relapse .
theophylline synergizes with ra / as2o3 therapy in a multirelapsed patient with ra / as2o3-resistant apl .
( a ) schematic representation of clinical events . ( b and c ) representative images of bone marrow samples taken at days 0 , 14 , and 28 of the initial ra / as2o3 course ( b ) or the following ra / as2o3/theophylline course ( c ) . note the rapid differentiation in ( c ) with the reappearance of normal erythroblasts at day 14 , as well as the maintenance of normal hematopoiesis despite leukemia relapse .
it is well known that camp greatly enhances the ra - induced differentiation of many cell lines derived from embryonal carcinoma or myeloid leukemias , including apl ( 10 ) . as low concentrations of as2o3
were found to induce only incomplete differentiation in an apl cell line ( 7 ) , we tested the hypothesis that camp would also enhance as2o3-induced differentiation .
even very low doses of as2o3 combined with 8-cpt - camp ( a low toxicity camp analog ) induced nbt reduction in 40% of the nb4 cells , whereas camp or as2o3 alone did not have significant effects ( unpublished data , see fig .
similarly , only combined as2o3 and 8-cpt - camp induced morphologic differentiation of nb4 cells into myelocyte - like cells ( unpublished data ) , as reported recently ( 17 ) .
to assess the possible in vivo efficiency of camp , we used transplantation apl model ( 14 ) derived from ra - sensitive pml / rara transgenic mice ( 18 ) .
we developed a similar transplantation model for ra - resistant apl , using leukemic cells from pml / rara transgenic mice in which a point mutation in the transgene impairs the binding of ra to pml / rara ( 16 ) .
in vivo growth of this leukemia is much slower than in the ra - sensitive model , liver , or spleen invasion is not as pronounced and either ra or as2o3 have mild antiproliferative effects in the absence of significant differentiation ( see fig .
mice bearing established leukemias were treated with 8-cl - camp , as2o3 , ra , or combinations of these drugs and killed 17 d after treatment .
continuous 8-cl - camp infusions allowed significant plasma concentrations to be reached ( 1 m on an average at day 3 ) . despite its toxicity , 8-cl - camp induced major antileukemic effects in both ra - sensitive and ra - resistant apls , as assessed by spleen weight ( fig .
myeloid cells undergoing differentiation were always observed in the marrow of ra - resistant apl after 7 d of treatment , and were often found upon treatment of ra - sensitive apl ( fig .
1 ) . in leukemic cells infiltrating the liver , a sharp reduction in the number of mitoses was observed after 8-cl - camp treatment ( 3% post- versus 28% pretreatment ) and a few condensed nuclei were seen ( unpublished data ) .
these results show that in these ra - sensitive or ra - resistant mouse models of apl , camp triggers a combination of cell growth arrest , differentiation , and apoptosis , leading to dramatic regressions of the leukemia .
8-cl - camp induces growth arrest in ra - sensitive ( ra , a ) or ra - resistant ( ra , b ) apl in mice . ( top )
spleen weight after 7 d of 8-cl - camp treatment or no therapy (  ) .
( bottom ) liver from the same mice . since camp greatly increased as2o3-triggered differentiation ex vivo in the nb4 cell line ( fig . 2    a )
, we combined 8-cl - camp and as2o3 treatments in vivo . with this combination ,
the spleen , liver , and bone marrow of mice with ra - sensitive apl became free of leukemia between days 1 and 3 , whereas mice treated with as2o3 or camp alone retained a significant tumor burden consisting of differentiating leukemic cells ( fig .
note that erythroblasts and megakaryocytes were extremely numerous in a cell - rich marrow ( fig .
2 e ) , in agreement with the idea that camp promotes the regrowth of these cells . in keeping with our ex vivo results , camp synergized with as2o3 to trigger differentiation , as shown by cd11b expression on bone marrow blasts at day 1 ( fig .
2 c ) , strongly suggesting that enhanced differentiation of the leukemic blasts contributes to these accelerated remissions .
the level of the cdk inhibitor p21 , a known camp target implicated in growth arrest , differentiation , and apoptosis , rose strikingly in bone marrow apl blasts upon treatment in vivo ( fig .
synergy was also noted between 8-cl - camp and ra with respect to both spleen weight and marrow cell differentiation , although this synergy was dimmed by the stronger differentiating effect of ra ( fig .
3 )   . arsenic and camp synergize to induce tumor regression and differentiation in ra - sensitive apl .
( a ) 8-cpt - camp potentiates as2o3-induced nbt reduction at 4 d in the apl cell line nb4 .
( b ) in a mouse model of apl , continuous infusion of 8-cl - camp for 3 d greatly reduces spleen weight and synergizes with as2o3 ( as ) to induce tumor reduction ( one representative experiment ) .
( c ) cd11b expression on bone marrow cells after 24 h of in vivo treatments , as indicated .
( d ) 8-cl - camp induces p21 expression in leukemic bone marrow after 24 h of in vivo treatment .
3 d of combined treatment restores normal hematopoiesis in the bone marrow ( e ) and induces tumor clearance from the liver ( f ) .
( g ) theophylline ( t ) therapy ( 3 d ) induces blast differentiation .
( a ) spleen weight after 7 d of treatment or untreated (  ) .
8-cl - camp induced even more dramatic regressions in ra - resistant apl , with some morphologically complete clearances ( figs . 1 and 4 )   .
unexpectedly , major enhancements of leukemia clearance and differentiation were always observed when ra was combined with 8-cl - camp ( fig .
although as2o3 alone triggered a slight antiproliferative effect in the absence of significant differentiation , the ra / as2o3 combination triggered minor , but reproducible , differentiation ( fig .
the stable camp derivative used here induces massive diuresis , precluding any long - term use , and may be further metabolized into potentially cytotoxic nucleotide analogs ( 15 ) . to ensure that the antileukemic effect indeed resulted from the activation of camp signaling , we used theophylline , a phosphodiesterase inhibitor which stabilized pools of endogenous intracellular camp . like 8-cl - camp ,
the - ophylline blocked growth in ra - sensitive apl and induced some apoptosis , accompanied by non - terminal differentiation ( fig .
as expected , the enhancement of differentiation was more pronounced with as2o3 than with ra ( fig .
however , there was no obvious boost in normal hematopoiesis , possibly reflecting low production of endogenous camp in normal cells .
on the basis of these promising results in mouse apl , an ra / as2o3-resistant apl patient was offered an experimental course of combined ra / as2o3/theophylline therapy .
previously , a month of the ra / as2o3 combination had led to a slow decrease in bone marrow blasts , a peak of differentiating myeloid cells in the blood ( fig .
5 )   , but normal hematopoiesis was not restored and the leukemia had reappeared 5 wk later ( fig . 5 , a and b ) . however , with combined ra / as2o3/theophylline treatment , leukemic cells underwent differentiation and normal erythroblasts appeared rapidly ( fig . 5 , a and c ) .
the patient no longer required red cell or platelet transfusions and white blood cells and hemoglobin reached levels close to the normal ones .
the patient remained leukemia free for 4 mo , but then the leukemic clone reappeared .
paradoxically , with ongoing ra / as2o3/theophylline therapy and despite the leukemia relapse , normal hematopoiesis has been maintained to date ( 5 mo since relapse ) , an extremely unusual situation in apl , in which cytopenia is the first sign of relapse .
theophylline synergizes with ra / as2o3 therapy in a multirelapsed patient with ra / as2o3-resistant apl .
( a ) schematic representation of clinical events . ( b and c ) representative images of bone marrow samples taken at days 0 , 14 , and 28 of the initial ra / as2o3 course ( b ) or the following ra / as2o3/theophylline course ( c ) . note the rapid differentiation in ( c ) with the reappearance of normal erythroblasts at day 14 , as well as the maintenance of normal hematopoiesis despite leukemia relapse .
our data demonstrate that in vivo activation of camp signaling was beneficial in two distinct mouse models of apl , as well as in an ra / as2o3-resistant apl patient .
the relative balance between growth arrest , apoptosis , and differentiation presumably depends on the dose of 8-cl - camp , the microenvironment of the apl blasts ( marrow versus liver metastasis , for example ) and their nature ( ra - sensitive or ra - resistant cells ) .
camp - triggered growth arrest may result from induction of the cdk inhibitor p21 , which was previously found to be involved in ra - induced apl cell differentiation ( 19 ) .
enhancement of ra- , as2o3- , or rexinoid - triggered differentiation by camp may also result from induction of g - csf expression ( 20 ) .
in f9 embryonal carcinoma cells , camp was shown to modulate ra - triggered differentiation directly through rara phosphorylation ( 21 ) .
since rara plays a critical role in myeloid differentiation , including in its regulation by il-3 and gm - csf ( 22 ) , the camp triggered myeloid differentiation may similarly be caused by modulation of rara signaling . at present
, we can not explain the greater sensitivity to 8-cl - camp of ra - resistant apl cells , but it might be due to their slower growth rate . in european trials , therapy - resistant patients only exceptionally exhibit mutations in pml / rara , which does not favor a direct parallel between the case of this patient and ra - resistant apl mice .
indeed , a cell line ( nb4-lr2 ) that harbors a mutation in pml / rara very similar to the one present in the ra - resistant apl cells used here , failed to differentiate upon combined ra/8-cl - camp treatment , but only matured upon combined camp / rexinoid exposure ( 20 ) . therefore
, our observations might reflect an in vivo metabolism of ra to rexinoids and activation of this parallel pathway .
absence of major synergy between camp and as2o3 in ra - resistant apl , compared to ra - sensitive disease , may reflect the low potency of as2o3 and the striking efficacy of 8-cl - camp in this setting .
as2o3 was initially believed to trigger apoptosis , but recent observations have suggested that differentiation was the dominant mechanism ( for a review , see reference 23 ) . the dramatic enhancement of as2o3-triggered differentiation by 8-cl - camp both ex vivo and in vivo ( fig .
2 and reference 17 ) , greatly strengthens this idea . in the patient , as in the apl mice , camp induced rapid regrowth of normal erythroblasts and megakaryocytes .
this might have resulted from a direct positive effect on the normal progenitors , as reported ex vivo ( 24 ) .
alternatively , apl cells might secrete inhibitors of normal hematopoiesis ( 25 , 26 ) whose synthesis or downstream signaling , may be blocked by camp . in the clinical setting
, such an in vivo stimulatory effect on normal hematopoiesis could be as important as leukemia inhibition .
the low toxicity of theophylline , its ability to accelerate ra- or as2o3triggered remissions and the observation that the rapid induction of ex vivo differentiation is predictive of outcome in apl ( 27 ) , could favor the use of theophylline for de novo apl patients .
in addition , unlike ra and as2o3 , camp does not obviously target pml / rara and might therefore be beneficial in malignancies other that apl .
indeed , patients with chronic lymphocytic leukemia experienced faster clinical remissions when theophylline was combined with low - dose chemotherapy ( 28 ) .
similarly , a patient on viagra , another phosphodiesterase inhibitor , exhibited regression of b cell chronic lymphocytic leukemia ( 29 ) .
many aml - derived cell lines are very sensitive to camp - triggered differentiation , particularly in the presence of other differentiation inducers . like histone desacetylase inhibitors , which unravel ra - induced differentiation ( 3 , 30 ) , the - ophylline
 differentiation therapy for acute myeloid leukemia uses transcriptional modulators to reprogram cancer cells . 
 the most relevant clinical example is acute promyelocytic leukemia ( apl ) , which responds dramatically to either retinoic acid ( ra ) or arsenic trioxide ( as2o3 ) . in many myeloid leukemia cell lines , cyclic adenosine monophosphate ( camp ) 
 triggers growth arrest , cell death , or differentiation , often in synergy with ra . 
 nevertheless , the toxicity of camp derivatives and lack of suitable models has hampered trials designed to assess the in vivo relevance of theses observations . 
 we show that , in an apl cell line , camp analogs blocked cell growth and unraveled as2o3-triggered differentiation . 
 similarly , in ra - sensitive or ra - resistant mouse models of apl , continuous infusions of 8-chloro - cyclic adenosine monophosphate ( 8-cl - camp ) triggered major growth arrest , greatly enhanced both spontaneous and ra- or as2o3-induced differentiation and accelerated the restoration of normal hematopoiesis . 
 theophylline , a well - tolerated phosphodiesterase inhibitor which stabilizes endogenous camp , also impaired apl growth and enhanced spontaneous or as2o3-triggered cell differentiation in vivo . 
 accordingly , in an apl patient resistant to combined ra  as2o3 therapy , 
 theophylline induced blast clearance and restored normal hematopoiesis . taken together , these results demonstrate that in vivo activation of camp signaling contributes to apl clearance , independently of its ra - sensitivity , thus raising hopes that other myeloid leukemias may benefit from this therapeutic approach . 
Introduction
Materials and Methods
Results
cAMP Synergizes with As
Antileukemic Effects of cAMP in Two Mouse Models of APL.
Theophylline Induces Remission in an RA- and As-resistant APL Patient.
Discussion