Abstract
Nucleophosmin 1 (NPM1) is a nucleus-cytoplasmic shuttling protein which is predominantly located in the nucleolus and exerts multiple functions, including regulation of centrosome duplication, ribosome biogenesis and export, histone assembly, maintenance of genomic stability and response to nucleolar stress. NPM1 mutations are the most common genetic alteration in acute myeloid leukemia (AML), detected in about 30–35% of adult AML and more than 50% of AML with normal karyotype. Because of its peculiar molecular and clinico-pathological features, including aberrant cytoplasmic dislocation of the NPM1 mutant and wild-type proteins, lack of involvement in driving clonal hematopoiesis, mutual exclusion with recurrent cytogenetic abnormalities, association with unique gene expression and micro-RNA profiles and high stability at relapse, NPM1-mutated AML is regarded as a distinct genetic entity in the World Health Organization (WHO) classification of hematopoietic malignancies. Starting from the structure and functions of NPM1, we provide an overview of the potential targeted therapies against NPM1-mutated AML and discuss strategies aimed at interfering with the oligomerization (compound NSC348884) and the abnormal traffic of NPM1 (avrainvillamide, XPO1 inhibitors) as well as at inducing selective NPM1-mutant protein degradation (ATRA/ATO, deguelin, (-)-epigallocatechin-3-gallate, imidazoquinoxaline derivatives) and at targeting the integrity of nucleolar structure (actinomycin D). We also discuss the current therapeutic results obtained in NPM1-mutated AML with the BCL-2 inhibitor venetoclax and the preliminary clinical results using menin inhibitors targeting HOX/MEIS1 expression. Finally, we review various immunotherapeutic approaches in NPM1-mutated AML, including immune check-point inhibitors, CAR and TCR T-cell-based therapies against neoantigens created by the NPM1 mutations.
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Introduction
Nucleophosmin 1 (NPM1) is a ubiquitous nucleus-cytoplasmic shuttling protein [1] predominantly resident in the nucleolus whose functions are pivotal for many cellular processes including histone assembly, centrosome duplication, ribosome biogenesis and export, maintenance of genomic stability and response to nucleolar stress [2].
Mutations of NPM1 gene are the most frequent genetic lesion in acute myeloid leukemia (AML), being detectable in about one-third of adult AML and 50–60% of AML with normal karyotype [3, 4]. These mutations are a driver genetic lesion and AML defining event that occurs in the context of clonal hematopoiesis, frequently promoted by genes such as DNMT3A and TET2 [4]. Distinctive features of NPM1-mutated AML include the mutual exclusion with recurrent cytogenetic abnormalities, the association with specific gene expression [5] and microRNA [6] profiles and the high stability of NPM1 mutations at relapse [7]. Another characteristic of NPM1-mutated AML is the aberrant cytoplasmic dislocation of the NPM1 mutant and NPM1 wild-type proteins (through heterodimerization) [3, 8, 9]. Moreover, the mutant NPM1 is directly involved in promoting high expression of homeobox (HOX) genes [10] which are necessary for maintaining the undifferentiated state of leukemic cells. Notably, this function is closely dependent on the cytoplasmic localization of the mutant. However, the mechanisms underlying leukemogenesis in NPM1-mutated AML still remain largely unknown [4]. According to the 5th edition of the World Health Organization (WHO) of hematolymphoid tumors, NPM1-mutated AML can be diagnosed irrespective of the percentage of blasts, based on previous observations that cases classified as MDS or MDS/MPN with NPM1 mutations quickly progressed to AML [11]. Because of the above unique features, NPM1-mutated AML is recognized as a distinct entity, within the category of AML with recurrent genetic abnormalities of the WHO classification [11]. The main characteristics of NPM1-mutated AML are summarized in Table 1.
The standard therapy of NPM1-mutated AML in young adults is based upon induction chemotherapy (±FLT3 inhibitors) and consolidation cycles with high/intermediate dose of cytarabine (ARA-C) ± allogeneic hematopoietic stem cell transplantation (HSCT) in first complete remission (CR), depending on the status of the FLT3 gene and measurable residual disease-MRD [12, 13]. However, despite the remarkable advances in the treatment of NPM1-mutated AML, about 50% of patients still died of progressive disease. Thus, there is a need for new therapeutic opportunities. Whole genomic approaches have unraveled the molecular heterogeneity of AML [14, 15] and given a great input to the development of small molecules aimed to target specific genetic abnormalities [16, 17]. Because of their small-size (<500 Da), these compounds can easily penetrate the cell membrane and exert their activity on intracellular proteins involved in cell signaling mechanisms (e.g., kinases) that promote the tumor growth [68], NPM1-mutated AML patients appear to be particularly sensitive to venetoclax (Fig. 2). Whether this efficacy is related to the high expression of HOX genes [5], that in turn are linked to BCL-2 inhibitor sensitivity and responsiveness [69], remains to be defined. NPM1 mutant-primed defect in mitochondrial function may also be responsible for the higher sensitivity to venetoclax [44].
NPM1-mutated AML can be targeted with selective inhibitors of menin to downregulate HOX/MEIS, with BCL-2 (venetoclax) to induce apoptosis and with inhibitors of the SYK pathway (entospletinib).
NPM1-mutated AML particularly benefits from venetoclax-based regimens, both at first diagnosis [70, 71] (independently by the FLT3 status [72]) and in the relapsed/refractory setting [4]. Alternatively, recruitment of NPM1 mutants to HOX loci through their interaction with chromatin-bound XPO1 [91] could be responsible for HOX expression maintenance, cytoplasmic delocalization of NPM1 representing a mere epiphenomenon. We previously suggested that NPM1 mutants may induce leukemia by acting both at the chromatin level and by delocalizing NPM1-interacting partners in the cytoplasm [4].
Dependence of NPM1-mutated AML cells on epigenetic machinery for HOX regulation [92] provides the rationale for using inhibitors of KMT2A-menin protein interaction [93, 94] (Fig. 2; Table 2). The VTP-50469 inhibitor showed strong in vitro and in vivo anti-leukemic activity on NPM1-mutated AML cells, causing a dose-dependent reduction in cell proliferation, a significant downregulation of HOXA/B clusters and MEIS1 gene expression, a marked differentiation of leukemic cells, a reduction of AML engraftment and a prolonged survival in mice PDX models [92, 95, 96]. This occurred especially through rapid repression of important co-factors of HOX genes (MEIS1 and PBX3), the effect on expression of HOXA and HOXB genes being not relevant [97]. The MI-3454 inhibitor was very effective in inhibiting cell proliferation and differentiation of NPM1-mutated AML cells, independently from the coexistence of other mutations in patients’ samples, and in reducing blast infiltration of organs and expression of MEIS1 and FLT3 in PDX NPM1-mutated mouse models [95].
Menin inhibitors were reported to prevent the transformation of Npm1-mutated mouse hematopoietic progenitors into leukemic cells, implying that they could also be effective in Npm1-mutated preleukemia [96]. However, we believe that this concept is difficult to translate into clinic because NPM1 mutations in patients do not associate with a preleukemic state [4]. Moreover, myelodysplasia with NPM1 mutations is very rare and cases with these characteristics usually represent already early-stage AML [12]. Thus, we expect that the value of menin inhibitors in clinic will be mainly limited to the therapy of frank NPM1-mutated AML.
Menin inhibitors was also combined with inhibitors of FLT3 (mutated in about 40% of NPM1-mutated AML [3]), demonstrating a synergistic effect that resulted in stronger cell growth inhibition, apoptosis and differentiation of AML blast cells [98] and induction of long-lasting CR in PDX mice models of NPM1-mutated/FLT3-ITD AML [99]. Menin inhibitors have been also combined with XPO1 inhibitors. The rationale for this association is that nuclear relocation of the NPM1 mutant by the XPO1 inhibitors is associated with downregulation of HOX genes [10]. Thus, the two compounds could exert a cumulative effect on downregulation of HOX genes through different mechanisms. The utility of combining menin inhibitors with venetoclax has been already mentioned [83]. All these associations could help preventing the rapid development of resistance to targeted monotherapies.
Menin inhibitors, including KO-539, SNDX-5613, JNJ-75276617 and DS-1594b, are currently evaluated in clinical trials [100, 101]. KO-539 induced CR in 2/6 patients with R/R AML who were evaluable for efficacy analysis. One of them had an NPM1-mutated AML co-mutated for DNMT3A and KMT2D who received KO-539 at 200 mg/die, as the eight line of therapy, achieving an MRD-negative CR [101]. This trial continues to enroll patients with NPM1-mutated and KTM2A-rearranged AML on the doses of 200 and 600 mg (NCT04067336). SNDX-5613 (an analog of VTP-50469) was very active in PDX mouse models of NPM1-mutated AML, including animals remaining in CR 1 year after cessation of therapy [96, 102]. The safety and efficacy of SNDX-5613 in adult patients with R/R NPM1-mutated or KTM2A-rearranged AML is being evaluated in the AUGMENT-101 phase I/II trial (NCT04065399). Preliminary results of this study were released by Syndax in April 2021. By March 2021, the trial had enrolled in the phase 1 cohort, 43 patients (median age 54 years) who had received a median of 3 previous lines of treatment. The most common side effects (>5%) included QT prolongation (14%), differentiation syndrome (5%) and anemia (5%). The overall response rate in the 7 patients with NPM1-mutated AML was 29% (2/7). In kee** with the proposed mechanism of menin inhibitors, RNA-Seq analysis of the bone marrow samples from responding patients exhibited downregulation of MEIS and HOXA9 genes and upregulation of the differentiation antigens CD11b, CD14 and CD13. The phase 2 part of the trial is ongoing. JNJ-75276617 is a potent inhibitor of the binding between menin and KTM2A. Its safety and activity are being tested in NCT04811560 that enrolls AML patients harboring NPM1 mutations or KMT2A rearrangements. However, no data have been released so far. The safety and efficacy of DS-1594b menin inhibitor will be evaluated as single drug or in combination with azacytidine and venetoclax regimens in a phase 1/2 clinical trial (NCT04752163). To maximize the depth and durability of clinical response, the Biomea Fusion, Inc. has recently developed BMF-219, an orally bioavailable, potent and selective irreversible covalent menin inhibitor. Clinical trial with this compound is ongoing (NCT05153330).
Targeting SYK signaling
In a phase 1b/2 trial (NCT02343939), entospletinib, a selective oral inhibitor of the spleen tyrosine kinase (SYK) which is constitutively activated in AML promoting survival and proliferation [103], when combined with chemotherapy, was more active in patients with HOXA9/MEIS1 signature (as in NPM1-mutated AML) than in the whole patient population [104] (Fig. 2). These results suggest that the increased expression and activity of SYK protein is strictly dependent upon the deregulation of HOXA and MEIS genes [105]. Based on this evidence, FDA approved a phase 3 trial to assess the efficacy and safety of entospletinib in combination with chemotherapy in adult patients with newly diagnosed NPM1-mutated AML (NCT05020665).
Immunotherapy of NPM1-mutated AML
Ideally, any target antigen for AML immunotherapy should be expressed at high levels in the whole leukemic population, including leukemic stem cells, and to be absent or low expressed in normal hematopoietic cells and other tissues. Leukemia-associated antigens (e.g., CD33 and CD123) are usually strongly expressed in AML cells (especially in NPM1-mutated AML cells [106,107,108]) but can also be detected in normal hematopoietic stem cells and in extramedullary tissues (e.g., CD123 in endothelial cells). This limits their use as target antigens for immunotherapy because of potential off-target effects.
Conversely, leukemia-specific antigens deriving from altered proteins encoded by leukemogenic mutations (e.g., NPM1), are specifically expressed in malignant clones and therefore represent ideal targets. In particular, the NPM1 mutant neoantigen can be considered an ideal AML target for a number of reasons. First, NPM1 mutations are common driver, gate-keeper events [109], very stable at relapse [4], specific for AML and absent in normal tissues [110]. Second, the NPM1 mutated proteins are detectable in chemoresistant leukemic stem cells [108], making them possibly vulnerable to immune surveillance and eradication. Third, although >50 NPM1 mutations have been identified, the 4 bp frameshift insertion occurring in NPM1 mutant A is responsible for almost 80-85% of all mutations and more rare NPM1 mutations lead to the same amino acidic changes at NPM1 C-terminus. Fourth, the newly acquired amino acid C-terminus sequence of NPM1 mutant proteins is highly immunogenic in animals, eliciting specific antibodies. Fifth, the aberrant localization of the NPM1 mutant proteins in the cytoplasm of leukemic cells may favor their processing by the Human Leukocyte Antigen (HLA) MHC class I degradation pathway leading to HLA presentation and anti-cancer immune response. Indeed, using in silico analysis, we predicted that several peptides could bind to specific HLA class I molecules [111]. Sixth, specific autologous cytotoxic T-cell responses against NPM1 mutant peptides could be detected in NPM1-mutated AML patients [112,113,114,115]. These immune responses associated with molecular CR [116] and may explain the relatively favorable outcome of NPM1-mutated AML [117]. interestingly, NPM1-mutated AML sensitivity to T-cell immunity has been observed not only in the autologous but also in allogeneic setting. Although NPM1-mutated AML patients without FLT3-ITD has a good prognosis, those who underwent allogeneic HSCT showed a particularly long-term disease control [118], probably due to specific graft-versus leukemia effect. Moreover, polyspecific T-cell anti-leukemic responses, even against NPM1-mutated peptides, have been observed following preemptive donor lymphocyte infusions (DLIs) at molecular relapse after allogeneic HSCT [116]. Finally, the importance of eradicating the NPM1-mutated clone to achieve cure of AML is exemplified by the clinical observation of patients with NPM1-DNMT3A double-mutated AML after cessation of therapy. These cases, when achieve long-term molecular (MRD-negative) remission, are likely to be cured, even though the persistence of detectable copies of the DNMT3A mutant (indicating persistent clonal hemopoiesis) may expose them to a low risk of develo** a second AML [4].
Antibodies against CD33 and CD123
CD33 is expressed in all stages of myeloid differentiation [119] and it is detectable in most cases of AML, the expression levels being high in NPM1-mutated AML [106]. Thus, CD33 is an useful target for immunotherapy with an anti-CD33 monoclonal antibody conjugated with a DNA-damaging calicheamicin derivative (Gemtuzumab Ozogamicin-GO) (Fig. 4). In a metanalysis study [120], adding GO to chemotherapy showed a survival benefit for intermediate-risk cytogenetics and NPM1-mutated AML patients because of a reduced relapse risk. Similar results were reported by the ALFA0701 trial that also demonstrated the impact of GO in reducing NPM1-mut transcripts level [121]. Higher reduction of NPM1-mut transcript levels were also observed in the GO arm of the AMLSG study that translated into a lower cumulative incidence of relapse [122]. However, the AMLSG 09-09 Phase III Study failed to meet the early primary end point (event-free survival) due to higher early mortality in the GO arm [123]. Nevertheless, a significant clinical benefit was observed in females older than 70 years with NPM1-mutated/FLT3 wild-type genotype [123] in terms of both event-free-survival and cumulative incidence of relapse. Collectively, the above studies support the incorporation of GO into the frontline treatment of NPM1-mutated AML.
NPM1-mutated AML can be targeted using antibody-drug conjugates (e.g., gentuzumab ozogamicin, anti-CD33), immune check-point inhibitors, CAR and TCR-based adoptive T-cell therapies directed against NPM1 mutated epitope/HLA complex. CAR chimeric antigen receptor, TCR T-cell receptor.
CD123 is highly expressed in NPM1-mutated AML both at diagnosis and relapse (Fig. 4), the highest expression being observed in CD34+ CD38− leukemic cells [107]. Moreover, CD123 expression was enhanced by FLT3 mutations, suggesting that the subset of NPM1/FLT3 double-mutated AML patients could particularly benefit from anti-CD123 targeted therapies [107]. So far, tagraxofusp (SL401) which is formed by the fusion of IL-3 with diphtheria toxin and the CD123-directed chimeric antigen receptor (CAR) T cells (MB-102) developed by Mustang Bio Inc. are approved or received the Orphan drug designation (MB-102) for the treatment of blastocytic plasmacytoid dendritic cell neoplasm [124] which strongly expresses CD123. Only scarce information is available in CD123-positive R/R AML. Main limitation of these products is myelotoxicity [124].
Antibodies against PD-1 and PD-L1
AML is a poorly immunogenic and highly immune-suppressive hematological malignancy. High expression of PD-L1 has been found in NPM1-mutated AML patients, especially in the leukemic progenitors/stem cell compartment (CD34+ CD38−) [125]. High PD-L1 expression in blasts of AML with NPM1-mutated/FLT3-ITD genotype predicted inferior survival [41]. Moreover, in a comprehensive immunogenomic analysis of AML, mutations of NPM1 and FLT3 preferentially associated with low T-cell cytolytic activity and a reduced expression of HLA-II (and/or related genetic determinants of HLA-II expression, as CIITA) [126]. Interestingly, CIITA methylation may limit antigen presentation by primary NPM1mutIDH1mut AML blasts through downregulation of MHC-II, thereby inducing immune evasion [126]. Immune evasion in NPM1-mutated AML is also contributed by VISTA (V-domain Ig suppressor of T-cell activation) and ULBP1 (NKG2 ligand) immunoregulatory circuitries that are both significantly upregulated in NPM1-mutated AML patients [126] (Fig. 4). VISTA-Ig, which shows a partial homology with other B7 family members, is predominantly expressed in hematopoietic cells of myeloid lineage. This circuitry in mainly involved in suppressing proliferation of T cells and blunting the production of T-cell cytokines, making it a potential target for immune check-point blockade combinations [127].
More recently, the anti-PD-1 antibody, nivolumab, was found to increase leukemia-associated antigen-stimulated cytotoxic T cells and cytotoxicity against stem cell-like cells, especially those carrying NPM1 mutations [128]. These findings provide a rationale for the treatment of NPM1-mutated AML, combining anti-PD-1 and anti NPM1-mutation specific immunotherapy (see below). Moreover, targeted immune gene expression and multiplexed digital spatial profiling showed distinct AML immune microenvironments [129]. The immune-infiltrated microenvironment that was characterized by severe immune suppression (high expression of PD-L1, CTLA4, IDO1 and BTLA), higher dependence from IFN-γ driven adaptive immune responses, high T-cell infiltration and expression of major histocompatibility complex, closely clustered with the adverse-risk genetic AML categories (specifically, TP53 and RUNX1 mutated AML) [129]. Conversely, the NPM1-mutated cases (with or without FLT3-ITD) more frequently showed an immune-depleted microenvironment [129]. Finally, NPM1-mutated AML harboring concomitant clonal hematopoiesis driven mutations (e.g., DNMT3A, TET2) showed an enriched tumor inflammation signature score, predicting a clinical benefit from anti-PD-1 treatment [130]. This finding is in kee** with the observation linking in a mouse model a persistent immune stimulation to an accelerated NPM1mut myeloproliferative phenotype in vivo [131].
Although the above findings suggest that NPM1-mutated AML may be a potential candidate for immune check-point inhibition (Fig. 4), the few studies performed so far with anti-PD-L1 antibodies in AML patients have shown only modest clinical activity. Their impact has been evaluated also in combination with hypomethylating agents [132,133,134], since they induce the expression of several immune-related genes, including HLA-I and HLA-II, leukemia-associated antigens (e.g., PRAME, WT1) [135], PD-1 and PD-L1 [136]. Despite most studies did not specifically evaluate NPM1-mutated AML, they showed that patients who might benefit more from these drug combinations are those who are naïve for hypomethylating agents or have <20% blasts and a higher pre-therapy infiltration of bone marrow by CD3+, CD4+ Teff, and CD8+ T cells [132]. The clinical trial NCT03769532 is currently evaluating the safety/efficacy of pembrolizumab plus 5-azacitidine in NPM1-mutated AML patients. The impact of pembrolizumab 200 mg (i.v. on day 14) has been assessed also in association with high-dose cytarabine in 37 R/R AML patients (9/37, 24%, bearing NPM1 mutations) [137]. The overall response rate, composite CR rate and median OS were 46%, 38% and 11.1 months, respectively. Responding patients exhibited a higher percentage of progenitor exhausted TCF1+ CD8+ T cells and an increased diversity of the T-cell receptor at baseline [137].
As previously mentioned, venetoclax is very active in NPM1-mutated AML [70]. This effect is also contributed by the immunomodulatory effect of the drug that enhances the T-cell-mediated anti-leukemic response by increasing reactive-oxygen species (ROS) production [138] and increases the PD1+ T-effector memory cells and anti-tumor efficacy in combination with immune check-point blockade [139]. The NCT02397720 trial is assessing the combination of nivolumab, azacytidine and venetoclax in frontline and R/R AML, while the NCT04284787 trial is assessing the impact of pembrolizumab plus azacytidine and venetoclax in newly diagnosed AML patients unfit for conventional chemotherapy.
CAR and TCR engineered T cell therapy
CAR T cells or T-cell receptor (TCR) gene therapy could be promising approaches against NPM1-mutated AML. Immune targeting can be distinguished into: (1) HLA-dependent therapies relying on the presentation of NPM1 neoantigen [113]; or (2) HLA-independent therapies identifying molecules differentially expressed on leukemic cells relative to normal cells (tumor-associated antigens).
Searching for HLA class I ligandome of primary AMLs, multiple ΔNPM1-derived immunogenic peptides, have been identified, including AIQDLCLAV, AIQDLCVAV, CLAVEEVSL, LAVEEVSLR, AVEEVSLRK 9-mer, and CLAVEEVSLRK 11-mer, representative of the more common NPM1 mutation types. These peptides are able to efficiently bind to at least most common HLA types (A*02:01, A*03:01) which are often detected in the Caucasian population [111,112,113,114,115, 140,141,142]. Using yeast surface display, a human single-chain variable fragment (scFv) that specifically identifies the NPM1 mutant epitope/HLA-A2 complex but not HLA-A2 or HLA-A2 loaded with control peptides was generated and used to construct CAR T cells (Fig. 4). These engineered cells showed strong in vitro and in vivo activity against preclinical models of NPM1-mutated AML cells carrying NPM1-mutant/HLA-A2 complex but not against NPM1 wild-type/HLA-A2+ AML cells or HLA-A2 negative tumor cells [140]. More recently, memory-like NK cells armed with the same neoepitope-specific CAR showed strong activity against NPM1-mutated AML in absence of toxicity [143].
CD123 and CD33 are strongly expressed both in NPM1-mutated AML cells and healthy tissue. Thus, aiming to improve selectivity for leukemic cells while minimizing toxicity towards normal cells, a dual targeting model was exploited through Cytokine Induced Killer (CIK) cells co-expressing a first-generation low affinity anti-CD123 and an anti-CD33 as costimulatory receptor without activation signaling domains. This trans-signaling strategy could allow: i) low toxicity profile against CD123+ endothelial cells and HSPC, due to a reduced cell activation given by the suboptimal first-generation CAR signal; ii) no or low myelotoxicity against CD33+ HSPC cells, due to absence of CIK cell activation upon the sole costimulatory signal engagement; and iii) full CAR-CIK activation only against double expressing CD123+/CD33+ leukemic cells [144] (Fig. 5).
The rationale of CD123/CD33 dual targeting trans-signaling strategy is to induce a full cell activation against only CD123/CD33+ leukemic cells while reducing cell stimulation against CD33+ HSPCs and CD123+ endothelial cells [144].
Specific T cells for HLA-A*02:01-binding CLAVEEVSL have been searched in healthy individuals using peptide-HLA tetramers. Tetramer-positive CD8+ T cells were isolated and their activity towards primary AMLs investigated. The TCR was then isolated from a clone with high anti-leukemic reactivity and its capability to specifically recognize and lyse HLA-A*02:01-positive ΔNPM1 AML demonstrated after retroviral transduction of CD8+ and CD4+ T cells [145]. Moreover, T cells transduced with TCR for HLA-A*02:01-binding CLAVEEVSL efficiently killed AML cells and prolonged OS of NSG mice engrafted with HLA-A*02:01-positive NPM1-mutated OCI-AML3 human cells [145] (Fig. 4). Thus, CLAVEEVSL is a neoantigen that can be efficiently targeted on AML by ΔNPM1 TCR gene transfer. While such TCR gene-engineered T-cell therapy prove to be potent and safe, it must match the TCR haplotype restriction to HLA-A*02:01-positive patients, representing around 40% of Caucasian population. Moreover, HLA class I downregulation or loss of neoantigen expression could be a possible mechanism of immune escape from NPM1-mutated TCR gene-engineered T-cell therapy.
DLI using T cells derived from healthy donors and specifically directed against the NPM1-mutated neoantigen with the aim to elicit graft-versus-leukemia may be a therapeutic option in patients experiencing molecular relapse following allogeneic HSCT. Eliciting endogenous immune responses through vaccination with NPM1 neoantigens is unlikely to be effective in patients with high-burden newly diagnosed or relapsed AML but could be of benefit, possibly in combination with immune check-point inhibitors, to treat pre-emptively NPM1-mutated AML with persistent NPM1 transcripts or in molecular relapse.
Future perspectives
Molecular mechanisms underlying NPM1-mutated AML are still poorly understood. Endogenous tagging of wild-type and mutated NPM1 proteins followed by mass spectrometry may unravel their interactions with other partners and functions in the cytoplasm. How NPM1 mutants deregulate the HOX program remains also to be better defined. Moreover, all current experimental data are based upon the analysis of OCI-AML3 and IMS-M2 human AML cell lines that both carry the most frequent NPM1 mutation (i.e., mutation A). Whether similar results can be extended also to the rarer NPM1 variants remains to be determined. Clarifying these issues may lead to the development of new targeted therapeutic strategies.
Unfit NPM1-mutated AML patients relapsing after venetoclax-based regimens represent a medical need. The mechanisms of resistance to venetoclax and its use in combination with other drugs to prevent relapse should be better investigated. Menin inhibitors are emerging as the most promising agents for targeted therapy of NPM1-mutated AML. The ongoing trials will tell us which is the real impact of these compounds in NPM1-mutated AML and suggest which are the best combinations to maximize the clinical benefit. Menin inhibitors, alone or in combination with venetoclax or other agents, could be incorporated in the treatment algorithm, as that shown in Fig. 3A, to reduce or eradicate NPM1-related MRD, possibly as bridge to allo-HSCT, in eligible patients. Moreover, menin and XPO1 inhibitors have the potential to be used alone or in combination (e.g., with FLT3 inhibitors), at initial diagnosis, especially in patients who are older or unfit for intensive chemotherapy.
Identifying novel drugs for NPM1-mutated AML using synthetic lethality approaches are under way [146]. High-throughput screening technology [147] allows to screen a large number of compound libraries at a rate that may exceed a few thousand compounds per day or per week [148, 149]. Searching for new molecules able to re-localize or reduce the expression of the NPM1 mutated protein, we have established a microscopy-based screening strategy suitable to analyze hundreds selected drugs and compounds using high-throughput microscope technique and image analysis (Fig. 6). Among other compounds, we identified inhibitors with known re-localizing activity on NPM1 mutated protein, thus confirming the value of our experimental strategy (unpublished data).
A Workflow of microscopy-based screening strategy (created with BioRender.com). B Example of 96 well plate subjected to image and data processing (generated with ShinyHTM software). Arrows indicate results obtained with XPO1 inhibitors (KPT-185, KPT-276 and KPT-330). The higher number of points for selinexor (KPT-330) results from its use as a positive control.
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Acknowledgements
This work has been supported by the European Research Council (ERC Adv grant 2016 n. 740230 to BF and ERC Cons grant 2016 n. 725725 to MPM) and the Italian Association for Cancer research (AIRC grant n. 23604 to BF and grant Start-Up 2019 n. 22895 to LB).
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BF had the original idea and wrote and supervised the work together with MPM and LB. RR, GP, SP, FM and IG contributed to the drug screening and to wrote the section of molecular therapeutic targeting of NPM1-mutated AML. SS take care of NPM1-mutated AML patients treated with venetoclax. VC followed the trial on actinomycin D in NPM1-mutated AML. VMP and AM contributed to write the section on immunotherapy of NPM1-mutated AML.
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MPM declares honoraria from Rasna Therapeutics, Inc for scientific advisor activities and serves as consultant for scientific advisory boards of Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer and Jazz Pharmaceuticals. LB declares consultancy at scientific advisory boards for Abbvie and Amgen. BF licensed a patent on NPM1 mutants (n. 102004901256449) and declares honoraria from Rasna Therapeutics, Inc for scientific advisor activities. The other authors declare no competing interests.
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Ranieri, R., Pianigiani, G., Sciabolacci, S. et al. Current status and future perspectives in targeted therapy of NPM1-mutated AML. Leukemia 36, 2351–2367 (2022). https://doi.org/10.1038/s41375-022-01666-2
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DOI: https://doi.org/10.1038/s41375-022-01666-2
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