Dactinomycin induces complete remission associated with nucleolar stress response in relapsed/refractory NPM1-mutated AML

Acute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies

CD123 Is Consistently Expressed on NPM1-Mutated AML Cells

NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) comprises about 30% of newly diagnosed AML in adults. Despite notable advances in the treatment of this frequent AML subtype, about 50% of NPM1mut AML patients treated with conventional treatment die due to disease progression. CD123 has been identified as potential target for immunotherapy in AML, and several anti-CD123 therapeutic approaches have been developed for AML resistant to conventional therapies. As this antigen has been previously reported to be expressed by NPM1mut cells, we performed a deep flow cytometry analysis of CD123 expression in a large cohort of NPM1mut and wild-type samples, examining the whole blastic population, as well as CD34+CD38− leukemic cells. We demonstrate that CD123 is highly expressed on NPM1mut cells, with particularly high expression levels showed by CD34+CD38− leukemic cells. Additionally, CD123 expression was further enhanced by FLT3 mutations, which frequently co-occur with NPM1 mutations. Our results identify NPM1-mutated and particularly NPM1/FLT3 double-mutated AML as disease subsets that may benefit from anti-CD123 targeted therapies

CD123 Is Consistently Expressed on <i>NPM1</i>-Mutated AML Cells

NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) comprises about 30% of newly diagnosed AML in adults. Despite notable advances in the treatment of this frequent AML subtype, about 50% of NPM1mut AML patients treated with conventional treatment die due to disease progression. CD123 has been identified as potential target for immunotherapy in AML, and several anti-CD123 therapeutic approaches have been developed for AML resistant to conventional therapies. As this antigen has been previously reported to be expressed by NPM1mut cells, we performed a deep flow cytometry analysis of CD123 expression in a large cohort of NPM1mut and wild-type samples, examining the whole blastic population, as well as CD34+CD38− leukemic cells. We demonstrate that CD123 is highly expressed on NPM1mut cells, with particularly high expression levels showed by CD34+CD38− leukemic cells. Additionally, CD123 expression was further enhanced by FLT3 mutations, which frequently co-occur with NPM1 mutations. Our results identify NPM1-mutated and particularly NPM1/FLT3 double-mutated AML as disease subsets that may benefit from anti-CD123 targeted therapies

NOVEL NPM1 EXON 5 MUTATIONS AND GENE FUSIONS LEADING TO ABERRANT CYTOPLASMIC NUCLEOPHOSMIN IN AML

none32siNucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but sporadically also exon 9 and 11, all causing changes at protein C-terminal end (loss of tryptophans and creation of a nuclear export signal-NES motif) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 AML patients, we found non-exon 12 NPM1 mutations in 5/387 (1.3%) NPM1c+ cases. Besides mutations in exon 9 (n=1) and exon 11 (n=1), novel mutations in exon 5 were discovered (n=3). One more exon 5 mutation was identified in additional 141 AML patients selected for wild-type NPM1 exon 12. Furthermore, 3 NPM1 rearrangements (i.e. NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13,979 AML samples screened by cytogenetic/FISH and RNA sequencing. Functional studies demonstrated that in AML cases the new NPM1 proteins harboured an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting any AML-associated NPM1 genetic lesions. Also, this study highlights the need for developing new specific assays for molecular diagnosis and monitoring of NPM1-mutated AML.noneMartelli, Maria Paola; Rossi, Roberta; Venanzi, Alessandra; Meggendorfer, Manja; Perriello, Vincenzo Maria; Martino, Giovanni; Spinelli, Orietta; Ciurnelli, Raffaella; Varasano, Emanuela; Brunetti, Lorenzo; Ascani, Stefano; Quadalti, Corinne; Cardinali, Valeria; Mezzasoma, Federica; Gionfriddo, Ilaria; Milano, Francesca; Pacini, Roberta; Tabarrini, Alessia; Bigerna, Barbara; Albano, Francesco; Specchia, Giorgina; Vetro, Calogero; Di Raimondo, Francesco; Annibali, Ombretta; Avvisati, Giuseppe; Rambaldi, Alessandro; Falzetti, Franca; Tiacci, Enrico; Sportoletti, Paolo; Haferlach, Torsten; Haferlach, Claudia; Falini, BrunangeloMartelli, Maria Paola; Rossi, Roberta; Venanzi, Alessandra; Meggendorfer, Manja; Perriello, Vincenzo Maria; Martino, Giovanni; Spinelli, Orietta; Ciurnelli, Raffaella; Varasano, Emanuela; Brunetti, Lorenzo; Ascani, Stefano; Quadalti, Corinne; Cardinali, Valeria; Mezzasoma, Federica; Gionfriddo, Ilaria; Milano, Francesca; Pacini, Roberta; Tabarrini, Alessia; Bigerna, Barbara; Albano, Francesco; Specchia, Giorgina; Vetro, Calogero; Di Raimondo, Francesco; Annibali, Ombretta; Avvisati, Giuseppe; Rambaldi, Alessandro; Falzetti, Franca; Tiacci, Enrico; Sportoletti, Paolo; Haferlach, Torsten; Haferlach, Claudia; Falini, Brunangel