Characterization of the clonal hierarchy and immunophenotype of PTPN11 mutations in acute myeloid leukemia.

Mutations in protein tyrosine phosphatase non-receptor type 11 ( PTPN11 ) have been considered late acquired mutations in acute myeloid leukemia (AML) development. To interrogate the ontogeny of PTPN11 mutations, we utilized single-cell DNA sequencing and identified that PTPN11 mutations can occur as initiating events in some AML patients when accompanied by strong oncogenic drivers, commonly NPM1 mutations. The co-driver role of PTPN11 mutations was confirmed in a novel murine model that exhibits an AML phenotype with early expansion of a diverse set of variably differentiated myeloid cells that engrafted into immunodeficient and immunocompetent mice. This immune diversity was reconstituted from early precursor cells when engrafted into immunodeficient mice. Moreover, immune diversity was also observed in the blast component of patient samples with NPM1 and PTPN11 mutations, providing novel antigen targets for immune based approaches in this subset of AML that is resistant to multiple targeted therapies.

INTRODUCTION: Noonan syndrome (NS) is an autosomal-dominant disorder that presents with a characteristic face, short stature, skeletal anomalies, and congenital heart defects. Protein-tyrosine phosphatase nonreceptor-type 11 (PTPN11), encoding SHP-2, mutation was the first reported gene involved and accounted for 31% to 60% of cases of NS. The KRAS gene was the second reported gene and was recently identified in a small number of patients with NS. OBJECTIVE: Our goal was to perform mutational analysis of PTPN11 and KRAS genes in children with NS. METHODS: In this study we screened for mutation of the PTPN11 and KRAS genes in 73 Taiwanese patients with NS. The mutation analysis of the 15 coding exons and exon/intron boundaries was performed by polymerase chain reaction and direct sequencing of the PTPN11 gene. The mutation analysis of 5 coding exons and exon/intron boundaries was performed by polymerase chain reaction and direct sequencing of the KRAS gene. We identified 12 different missense PTPN11 mutations in 15 (21%) patients with NS and 2 different missense KRAS (V14I and I36M) mutations in 2 (3%) patients with NS. These PTPN11 gene mutations were clustered in exon 3 (n = 6) encoding the N-SH2 domain and 13 (n = 5) encoding the PTP domain. CONCLUSIONS: This study provides support that PTPN11 and KRAS mutations are responsible for NS in Taiwanese patients.

Background: The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) has few cytotoxic T cells and a predominance of immunosuppressive cells, constraining its response to checkpoint inhibitors targeting PD-1/PD-L1 and CTLA4. We recently observed that targeting protein tyrosine phosphatase non-receptor type 22 (PTPN22), a negative immune regulator, reduces tumor growth by ∼80% in an immunotherapy-resistant model of PDAC when combined with TNFRSF costimulators 41BB or OX40. These observations prompted the interrogation of the interplay between PTPN22 and TNFRSF pathways, both in T cells and myeloid cells, to determine how immunosuppressive barriers are overcome. Methods: To evaluate the effects of genetic abrogation of PTPN22, mice constitutive or myeloid-specific conditional knock out (KO) of PTPN22 were inoculated with syngeneic KPCY 6419c5 or 6422c1 cells. For pharmacologic targeting of PTPN22, we employed our small molecule PTPN22 inhibitor, L1. To determine clinical relevance, we curated and analyzed the immune profiles of patients who are heterozygous for the dysfunctional PTPN22 SNP R620W (and matched wildtype (WT) controls) undergoing checkpoint immunotherapy using CyTOF. Results: We discovered that OX40 costimulation led to TRAF3 degradation, resulting in PTPN22 translocation from the cytosol to the membrane, likely facilitating further inhibition of T cell receptor activation. Deep profiling of tumors treated with anti-PD1 and OX40 agonism upon PTPN22 abrogation revealed that intratumoral T cell expansion was driven via promotion of CD4+ helper T cell function with significant upregulation of IFN-γ, TNF-α, and IFN-α pathways. Efficacy was reproduced with a 41BB agonist and validated in orthotopic PDAC tumor models and with administration of L1. Consistent with these preclinical findings, cancer patients with the PTPN22 R620W allele undergoing immunotherapy exhibited significantly increased OX40 and PD1 expression on T cells compared to WT, highlighting the co-regulation of these immunomodulatory pathways. Conclusion: Abrogation of PTPN22 enhances PDAC immunotherapy efficacy by significantly reducing tumor burden through increasing T cell infiltration when combined with anti-PD1 and TNFRSF costimulators (OX40 or 41BB). This approach promotes CD4+ T cell activation and upregulates key inflammatory pathways. Moreover, patients heterozygous for the PTPN22 R620W allele undergoing immunotherapy showed elevated OX40 and PD1 expression on T cells underscoring the translatability of our pre-clinical findings. Citation Format: Soren Charmsaz, Nicole E. Gross, George W. Apostol, Jae W. Lee, Jianping Lin, Alexei G. Hernandez, Erin M. Coyne, Sarah M. Shin, Courtney Cannon, Xuan Yuan, James M. Leatherman, Daniel J. Zabransky, Mark Yarchoan, Zhong-Yin Zhang, Elizabeth M. Jaffee, Won Jin Ho. PTPN22-based immunotherapy yields near-complete response against PDAC via multi-pronged immune modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6407.

Study Of Activity Of E6201, a Dual FLT3 and MEK Inhibitor, In Acute Myelogenous Leukemia With FLT3 Or RAS Mutation

Prognostic Impact of Molecular Mutations in AML at First Relapse

Normal-Karyotype Acute Myeloid Leukemia

DNA methyltransferase inhibitors (DNMTis), which transcriptionally activate hypermethylated genes in cancers and leukemias, also activate endogenous retroviruses (ERVs), leading to increased cytosolic double-stranded (ds) RNA and interferon (IFN) signaling, in a process termed viral mimicry. The tumor suppressor TP53 has been reported to cooperate with DNA methylation and IFN signaling to maintain transcriptional silencing of various repeat sequences, including ERVs. We now report that DNMTis, used to treat acute myeloid leukemia (AML) patients unfit for chemotherapy, robustly induce expression of ERVs and STING-dependent IFN/inflammasome signaling in AML with mutations in TP53(approximately 10%), a subset with poor prognosis. First, our studies of ERV transcripts (N=13) levels in TP53 mutant (KASUMI, KG1-1A, U937) and wild-type (WT; MOLM-14, OCI/AML2, and OCI/AML3) AML cell lines and primary cells (N=6 mutant, N=6 WT) show that, while TP53 mutant cells have low baseline ERV expression, DNMTi treatment significantly increases ERV expression, compared with WT TP53 cells (p<0.05). Moreover, inhibiting TP53 in WT TP53 AML cell lines with the specific TP53 inhibitor pifithrin significantly upregulates ERVs, validating the role of TP53 in suppressing ERVs in AML cells. Second, while TCGA data analysis showed that baseline STING transcript are low in TP53 mutant vs WT AML, phospho-STING (activated) is actually increased in TP53 mutant AML cells and DNMTi treatment further activates STING. Increased expression of downstream IFN/inflammasome genes in TP53 mutant AML treated with DNMTi, compared with results in WT cells, validates these results. Moreover, DNMTi combination with poly (ADP-ribose) polymerase inhibitors (PARPis), which we previously reported to activate STING in breast and ovarian cancer though increasing cytosolic dsDNA and dsRNA, further augments STING activation and IFN/inflammasome signaling (p<0.05). Third, the IFN/inflammasome signaling response to DNMTI/PARPI signaling is directly linked with decreased expression of DNA ds break repair (DSBR) genes and activity, leading to homologous recombination defects (HRD). Fourth, the mechanism for both IFN/inflammasome signaling and HRD induction is dependent on STING, since both CRISPR/CAS KO of STING or treatment with the STING inhibitor H-151 can rescue these effects in TP53 mutant and WT AML cells. Our findings increase understanding of the therapy potential for combining DNMTi+PARPi treatment in AML and suggest use of this paradigm to treat mutant TP53 AML. These results also suggest that activating STING may be a key strategy for increasing IFN/inflammasome signaling in treatment of TP53 mutant AML and that immune responses may be further augmented with immune therapy in AML and other cancers. Citation Format: Aksinija A. Kogan, Michael J. Topper, Lora Stojanovic, Lena J. McLaughlin, Tammy J. Kingsbury, Maria R. Baer, Michael Kessler, Stephen B. Baylin, Feyruz V. Rassool. DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6301.

Clinical characteristics and comutational landscape of CSF3R mutations in acute myeloid leukemia

Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) occur in about 15% of acute myeloid leukemia (AML) patients, and lead to the production of an oncometabolite, the 2-hydroxyglutarate (2-HG). Importantly, many clinical studies demonstrated that IDH1/2 mutations are systematically conserved at relapse, suggesting that this mutated enzyme could contribute to early leukemogenesis. In an effort to identify therapy specifically targeting IDH1 R132H mutated blasts, we developed an AML cell line model carrying IDH1 R132H mutation. We first stably transduced HL60 cell line to establish clones carrying either empty vector, IDH1 WT gene or IDH1-R132H mutated gene. We then characterized these clones with different features including 2-HG production. Since demethylation of many genes involved in the retinol metabolism is impaired by IDH1-R132H mutation and because all-trans retinoic acid (ATRA), a retinol metabolite, is already used in clinic, we studied its effects on IDH1-R132H mutated AML cells. Here, we showed on our transduced models as well as on primary AML patient samples (n=6), that AML blasts harboring IDH1-R132H mutation were highly sensitive to low doses of ATRA, compared to the IDH1 WT. In fact, IDH1-R132H mutation sensitized to ATRA-induced differentiation, as shown by increased expression of the differentiation marker CD11b and morphological changes evidenced with May-Grünwald-Giemsa staining. This was followed by changes of CD14 and CD15 expression, as well as granulocytic enzymatic activity measured with the nitroblue tetrazolium reduction assay. This terminal granulocytic differentiation came with the reduction of proliferation and colony formation, and then, quickly led to apoptosis of IDH1 R132H mutated AML cells. Moreover, we showed that the level of differentiation of IDH1 R132H mutated models was correlated to the magnitude of IDH1-R132H protein expression and thus to the 2-HG production. We further demonstrated that treatment with a cell-permeable form of 2-HG sensitized empty construct to ATRA-induced differentiation, whereas the inhibition of IDH1-R132H activity reduced its ATRA-sensitivity in IDH1-R132H mutant. Altogether, our results show that IDH1-R132H mutation sensitizes to ATRA-induced granulocytic terminal differentiation in a 2-HG concentration-dependent manner and that it leads to their apoptosis. Since serum 2-HG concentration of IDH mutated patients is approximately 100 fold higher than for non mutated patient, we could expect a high and specific effect of ATRA treatment on IDH1 mutated AML patients. Furthermore, as this mutation could contribute to relapse, this might be a promising therapeutic strategy to achieve a long-term remission in AML with IDH1 mutations. Citation Format: Helena Boutzen, Fabienne De Toni, Estelle Saland, Eric Delabesse, Véronique De mas, Cécile Demur, Florence Castelli, Lara Gales, Virginie Penard-lacronique, Stéphane De Botton, Jean-Charles Portais, Christophe Junot, Stéphane Manenti, Christian Récher, Jean-emmanuel Sarry. All-trans-retinoic acid as a new therapeutic approach to target isocitrate dehydrogenase mutations in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2678. doi:10.1158/1538-7445.AM2014-2678

The transcription factor CCAAT/enhancer binding protein-alpha (CEBPA) is crucial for normal myeloid differentiation. Mutations in the CEBPA gene are found in subsets of patients with acute myeloid leukemia (AML). Recently, three families were reported in whom several family members had germline CEBPA mutations and subsequently developed AML. Whereas familial AML is considered a rare event, the frequency of CEBPA germline mutations in AML is not known. In this study, we screened 187 consecutive AML patients for CEBPA mutations at diagnosis. We detected 18 patients (9.6%) with CEBPA mutations. We then analyzed remission samples and constitutive DNA from these patients. We found that two (11.1%) of 18 AML patients with CEBPA mutations carried a germline N-terminal frameshift CEBPA mutation. Interestingly, additional members in the families of both of these patients have been affected by AML, and the germline CEBPA mutations were also observed in these patients. Additional somatic mutations in AML patients with germline CEBPA mutations in the two families comprised in-frame C-terminal CEBPA mutations in two patients, two nonsilent CEBPA point mutations in one patient, and monosomy 7 in one patient. This study shows, for the first time to our knowledge, that germline CEBPA mutations are frequently observed among AML patients with CEBPA mutations. Including the families with germline CEBPA mutations reported previously, additional somatic CEBPA mutations represent a frequent second event in AML with germline CEBPA mutations. Our data strongly indicate that germline CEBPA mutations predispose to AML and that additional somatic CEBPA mutations contribute to the development of the disease.

Neurofibromin 1 gene Mutations Are Recurrent Events in Adult Patients with Acute Myeloid Leukemia (AML), Associate with Poor Outcome and Refine the Adverse European Leukemianet (ELN) Risk Category

Acute myeloid leukemia (AML) is the most prevalent form of leukemia among adults, characterized by aggressive behavior and significant genetic diversity. Despite decades of reliance on conventional chemotherapy as the mainstay treatment, patients often struggle with achieving remission, experience rapid relapses, and have limited survival prospects. While intensified induction chemotherapy and allogeneic stem cell transplantation have enhanced patient outcomes, these benefits are largely confined to younger AML patients capable of tolerating intensive treatments. DNMT3A, a crucial enzyme responsible for establishing de novo DNA methylation, plays a pivotal role in maintaining the delicate balance between hematopoietic stem cell differentiation and self-renewal, thereby influencing gene expression programs through epigenetic regulation. DNMT3A mutations are the most frequently observed genetic abnormalities in AML, predominantly in older patients, occurring in approximately 20–30% of adult AML cases and over 30% of AML with a normal karyotype. Consequently, the molecular underpinnings and potential therapeutic targets of DNMT3A mutations in AML are currently being thoroughly investigated. This article provides a comprehensive summary and the latest insights into the structure and function of DNMT3A, examines the impact of DNMT3A mutations on the progression and prognosis of AML, and explores potential therapeutic approaches for AML patients harboring DNMT3A mutations.

The Autoimmunity-Associated Gene PTPN22 Potentiates Toll-like Receptor-Driven, Type 1 Interferon-Dependent Immunity

Role of Epigenetic Readers in Pathogenesis and Therapy of Acute Leukemias

This issue of IJH contains five serial review articles on acute myeloid leukemia (AML). AML is a molecularly heterogeneous neoplasm, and the understanding of its molecular pathogenesis continues to grow. The development of chemotherapy and hematopoietic stem cell transplantation (HSCT) have increased 5-year survival rate to 40 % in AML patients younger than 60—65 years, whereas outcome in elderly AML patients remains dismal. In the first review, David Grimwade summarizes the molecular heterogeneity of AML and clinical significance of each molecular abnormality. In his work with the UK Medical Research Council (MRC), he has published important articles on prognostic factors in AML and characterization of the mechanisms underlying leukemiaassociated chromosomal translocations. A consensus had emerged that leukemogenesis requires two classes of mutations in AML (WHO classification 2008). The first of these is abnormalities in transcriptional factors that impair hematopoietic differentiation. Rearrangement of genes such as RUNX1, RARA, or MLL belongs to this class of mutations as well as point mutation of CEBPA (these are known as class II mutations). The second class of mutations consists of FLT3, KIT, or NRAS mutations (class I mutations), which are associated with cellular proliferation and/or survival. The discovery of the NPM1 mutation in 2007, however, suggested that the story is not so simple. From 2009, genome sequence analysis revealed novel gene mutations such as TET2, IDH1/2, and DNMT3A especially in AML showing normal karyotype. These gene products are associated with epigenetic modifications, including DNA methylation, and their biological significance in leukemogenesis is under investigation. In the second perspective, Martin Tallman, chairman of the Leukemia Committee for the Eastern Cooperative Oncology Group (ECOG), reviews induction chemotherapy for AML. Although the fundamental drug combination for remission-induction, DNR or IDA, plus Ara-C, has not changed for more than 30 years, there is still no definitive protocol for dose and schedule. Furthermore, many trials are under way seeking to improve CR rate and survival; these experimental approaches include intensification of anthracycline, high-dose of Ara-C, addition of a third drug, priming usage of G-CSF, and combination with molecular targeted drug. Third, co-chairman of the Japan Adult Leukemia Study Group (JALSG) Shuichi Miyawaki was invited to overview Japanese studies on AML. The JALSG has just celebrated its 25th anniversary with the holding of an international symposium at Tokyo Forum on June 23–24, 2012, which this review article commemorates. JALSG has completed six studies of AML to date. As Ryuzo Ohno previously reviewed the first four JALSG studies from 1987 to 1997 (Int J Hematol. 72:272, 2000), Miyawaki focused primarily on the two studies after 1997. Working with Masamitsu Yanada, I co-authored the fourth review, focusing on elderly AML, which is becoming a more important issue in many countries. In the JALSG data, the median age of newly diagnosed patients with AML and high-risk MDS is around 65 years. Accordingly, at least half of AML patients are not included in clinical studies using intensive chemotherapy and HSCT. Since conventional therapy has limitations in improving outcomes in elderly AML, new therapeutic approaches are urgently needed. T. Naoe (&) Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan e-mail: tnaoe@med.nagoya-u.ac.jp