Modulation of the sensitivity to ruxolitinib-mediated JAK2 inhibition by mutationally activated SHP2 exhibits cell context dependency in pre-clinical models of myeloproliferative neoplasms
The identification and characterization of an activating point mutation of SHP2 (encoded by the PTPN11 gene), SHP2-F71L, in UKE1 cells is reported, suggesting activating PTPN11 mutations have the potential to desensitize the effects of JAK2 inhibition therapy in patients undergoing therapy and may be dependent on unknown cell and molecular profile contexts.
Abstract
Classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are hematopoietic stem cell cancers that result in aberrant trilineage myeloid cell proliferation, bone marrow fibrosis, and increased risk of acute myeloid leukemia. MPNs are driven by deregulated activity of the JAK2 kinase, induced by mutations in the JAK2, CALR, and MPL genes, but approved JAK2 inhibitors primarily offer palliative effects, not remission. Cell models that demonstrate MPN oncogene driven JAK2 activity requisite for cell proliferation are important research tools for the development of anti-JAK2 and anti-JAK2 signaling therapeutics for MPN. SET2 and UKE1 cells are two such cell lines, as they express JAK2-V617F, one of the major driving mutations of MPN, and require signaling by JAK2 for their growth and viability. These cell lines are AML cell lines that were derived from patients with a previous diagnosis of MPN before they developed AML. Our previous studies demonstrated that the SHP2 phosphatase may be a therapeutic target for MPNs, and here we report our identification and characterization of an activating point mutation of SHP2 (encoded by the PTPN11 gene), SHP2-F71L, in UKE1 cells. Given SHP2 functions downstream of JAK2 and mediates JAK2 activation of RAS, we set out to determine the effect of mutational activation of SHP2 on the sensitivity of MPN model cells to JAK2 inhibition. We used CRISPR-Cas9 to edit this mutation in UKE1 cells back to wildtype such that these cells only express wildtype SHP2. These cells exhibited enhanced sensitivity to SHP2 inhibition and, notably, enhanced sensitivity to the JAK2 inhibitor ruxolitinib. This altered sensitivity was reverted by exogenous expression of SHP2-F71L but not SHP2-WT, indicating expression of an activated SHP2 may alter sensitivity to JAK2 inhibition in MPN model cells. We further explored this by genetically editing SET2 cells to express SHP2-F71L but observed no change in SHP2 inhibitor or JAK2 inhibitor sensitivity in cells with a SHP2-F71L encoding allele of PTPN11. Using the cytokine dependent BaF3 cell line where deregulation of JAK2 signaling by expression of JAK2-V617F induces cytokine independent transformation that remains dependent on this JAK2 signaling, we observed no effect of the expression of an activated SHP2 mutant on the sensitivity of the growth and viability of these cells to ruxolitinib. Recent studies have demonstrated activation of RAS signaling can antagonize JAK2 inhibition in pre-clinical MPN models, and the presence of RAS pathway mutations associates with patients whose disease advances on ruxolitinib therapy. Such mutations include activating mutations in PTPN11, as SHP2 is an upstream activator of RAS signaling. Our results suggest that activating PTPN11 mutations have the potential to desensitize the effects of JAK2 inhibition therapy in patients undergoing therapy and may be dependent on unknown cell and molecular profile contexts.
Myelofibrosis (MF) is a chronic myeloproliferative neoplasm (MPN) characterized by splenomegaly, constitutional symptoms, marrow fibrosis, cytopenias and inflammation. Janus kinase 2 (JAK2) inhibitors, such as ruxolitinib, reduce splenomegaly and alleviate symptoms but have limited disease-modifying activity, and resistance frequently develops. Selinexor, an oral selective exportin (XPO1) inhibitor, restores nuclear retention of tumour suppressors and inhibitors of inflammatory signalling. Dual XPO1/JAK2 inhibition targets complementary downstream pathways, enhancing suppression of MF progenitor cell fitness. We evaluated selinexor plus ruxolitinib in MF models, including JAK2V617F or calreticulin (CALR) exon 9-mutant MPN cell lines and samples from patients with MF. The combination showed greater anti-proliferative activity than JAK2 inhibition alone and suppressed colony formation from MF Cluster of Differentiation 34 (CD34)+ cells. Selinexor remained active in a ruxolitinib-resistant MPN cell line, inducing G1 arrest and apoptosis. Multi-omic analyses demonstrated increased nuclear retention of p53 and disruption of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling, complementing and extending ruxolitinib-targeted pathways. The combination suppressed NF-κB transcriptional activity and reduced Tumor Necrosis Factor alpha (TNFα), Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1) secretion from MF patient peripheral blood mononuclear cells. These findings highlight that selinexor plus ruxolitinib affects both MF haematopoietic cell-intrinsic and microenvironment-related pathways, providing a novel disease-modifying strategy with the potential to improve clinical outcomes in patients with MF.
T. Kashyap, Andrea A Ellero, C. Walker et al.· British Journal of Haematolo...· 0 citations
Myelo fi brosis (MF) is caused by expansion of mutated hematopoietic stem and progenitor cells that results in bone marrow (BM) fi brosis, ineffective erythropoiesis, elevated in fl am-matory cytokines, and extramedullary hematopoiesis. Most MF patients experience constitutional symptoms that impair quality of life. Cytopenias, thromboembolism, and leukemic transformation cause morbidity and mortality [1]. Aberrant JAK/STAT pathway activation by mutations in JAK2 , CALR , or MPL is central to MF pathogenesis, and JAK inhibitors (JAKis) are the standard of care. Ruxolitinib, the fi rst JAKi approved for MF, effectively reduces spleen volume and symptoms in ~40% of patients, but many responses are only transient [2]. Myelosuppression frequently necessitates dose reduction, compromising symptom control. While JAK2 V617F variant allele frequency (VAF) and fi brosis are reduced in some patients, JAKis do not eliminate the malignant clone, leaving patients at risk of progression. The limited ef fi cacy of JAKis has stimulated research into targeting other pathways involved in MF pathogenesis [3]. Although activity was observed for several agents, to-date, JAKis have remained the only FDA-approved MF drugs. Allogeneic stem cell transplant (SCT), while potentially curative, is associated with high morbidity and mortality, and many patients are ineligible [4]. We have previously identi fi ed exportin-1 (XPO1)-mediated nuclear-cytoplasmic export (NCE) as a vulnerability in MF cells [5]. Selinexor, a fi rst-in-class XPO1 inhibitor approved for multiple myeloma
S. Tantravahi, Andrea A Ellero, Ami B. Patel et al.· Blood Cancer Journal· 1 citation
Advances in the molecular characterization of acute myeloid leukemia (AML) in recent decades have driven the development and clinical introduction of several targeted therapies, and menin inhibitors are the most recent additions to the therapeutic arsenal of AML treatments. The KMT2A and NPM1 loci have been the focus of intense investigation over the past two decades because of their well-established roles leukemogenesis, particularly in pre-clinical models of AML involving human xenografts. The ability of KMT2A and NPM1 aberrancies to drive and sustain leukemogenesis has provided a strong rationale for the development of therapeutic strategies to disrupt these molecular pathways. Menin inhibitors preferentially eliminate clones harboring KMT2A rearrangements or NPM1 mutations due to the dependence of these leukemia cells on the menin–KMT2A complex and its downstream HOXA9/MEIS signaling to sustain leukemic self-renewal and disease maintenance. In this review, we discuss the biological rationale for menin–MLL disruption in patients with acute leukemia with KMT2A rearrangement or NPM1 mutation. We explore data from the landmark AUGMENT-101 and KOMET-001 trials, which have led to regulatory approval of revumenib and ziftomenib for select patients. We also explore cutting-edge studies using menin inhibitors in combination strategies, including data from KOMET-007. The evidence supporting combination approaches remains based on early-phase, single-arm trials with relatively short follow-up, and longer-term and comparative data are needed to define their clinical benefit. We highlight resistance mechanisms that have emerged from the use of contemporary menin inhibitors and efforts aimed at addressing this resistance, with a focus on emerging clinical data on enzomenib and bleximenib. Finally, we discuss prospects on the putative role of menin inhibitors in the measurable residual disease (MRD)-positive and maintenance settings in AML. Menin inhibitors have successfully transitioned from biological proof-of-concept to approved targeted therapy, and future advances will depend on rational front line combination strategies, MRD-guided treatment, post-transplant maintenance strategies, and therapeutic sequencing informed by mechanisms of resistance.
Tina Y. Zhang, Shyam A. Patel, T. Badar· Cancers· 0 citations
Background/Objectives: Revolutionary small molecule, targeted, tyrosine kinase inhibitors (TKIs) in the clinic have engendered the perception that optimal treatment has already been established for diseases like chronic myeloid leukemia (CML). Addressing BCR::ABL1 oncokinase domain mutations in CML is unlikely to redress disease recrudescence owing to persistent leukemia stem cells (LSCs). Therefore, it is necessary to consider an alternative strategic approach: disrupting LSC interactions with the protective microenvironment and/or immune system. The interaction of the TNF-α superfamily member, CD27, with its upregulated ligand, CD70, initiates survival signaling specifically in CML cells when BCR::ABL1 is inhibited by TKIs and thus represents an attractive target. Previous studies modulating the expression of CD27 on LSCs in a mouse model of advanced phase CML were encouraging. In our study, we explored the CD70/CD27 axis as a therapeutic target in early-phase disease. Methods: Primitive CD34+ cells from treatment-naïve chronic phase (CP) CML patients were drug-exposed in an in vitro co-culture system; combination drug treatments of the therapeutic anti-CD70 antibody and TKIs, nilotinib, were assessed in our CP CML murine model. Results: The blockade of the CD70/CD27 axis in combination with TKIs did not result in greater LSC elimination than with nilotinib as a single agent in our CP CML models. Nonetheless, there was an observed reduction in CD70+ cells with combination treatment. Conclusion: Further preclinical study of the antibody as an adjunct in CD70-expressing hematological malignancy is perhaps warranted.
Jennifer E. Cassels, M. Drotar, Alyson MacNeil et al.· Hematology Reports· 0 citations
Philadelphia chromosome-positive leukemias involve signaling systems beyond BCR-ABL1, and treatment response may depend on this wider network biology. Second-generation BCR-ABL1 tyrosine kinase inhibitors such as dasatinib, nilotinib, and bosutinib show variable clinical responses according to diagnosis and disease stage. Dasatinib is effective in this context because it is active against SRC-family kinases as well as receptor-proximal kinases and downstream outputs involving the MAPK/ERK, PI3K/AKT and JAK/STAT pathways. This broader pharmacological profile may impair compensatory signaling, which may partly account for its activity against CML and Philadelphia chromosome-positive ALL, especially if SRC-linked or ERK-mediated persistence underlies treatment escape. However, the same breadth could trigger pleural effusion and immune-related effects, underscoring the need for personalized dosing, toxicity monitoring, and adaptive interpretation of responses. This review combines many forms of evidence, including mechanistic, phosphoproteomic, immunologic and clinical evidence, to show that dasatinib acts as a network-modulating tyrosine kinase inhibitor rather than only as an ABL1-directed agent. Translational biomarkers, host immune responses, and systems-biology strategies may help predict incomplete pathway inhibition, guide treatment selection, and improve the durability of deep molecular remission.
S. Vanitha, R. Kothai, B. Arul· Journal of Applied Pharmaceu...· 0 citations
Cluster of differentiation 47 (CD47), a 'don't eat me' immune checkpoint receptor, is frequently overexpressed on multiple myeloma (MM) cells and contributes to immune evasion. While its immune regulatory function is well established, the intrinsic oncogenic role of CD47 remains poorly understood. Here, we demonstrate for the first time that CD47 intrinsically promotes myeloma cell proliferation and survival by activating the JAK/STAT3-MYC (Janus kinase/signal transducer and activator of transcription 3 - MYC) signalling axis. CD47 overexpression increased cell growth, accelerated G1-M-cell cycle progression and promoted anchorage-independent colony formation, accompanied by elevated phosphorylation of STAT3 and upregulation of MYC, whereas CD47 knockdown produced the opposite effects. Transcriptomic analysis of patient datasets revealed that high CD47 expression was associated with enrichment of the JAK-STAT3 and MYC pathways, and CD47 levels positively correlated with STAT3 and MYC expression, suggesting a potential regulatory interplay among these molecules. Pharmacological inhibition using the JAK1/2 inhibitor ruxolitinib (Rux) decreased cell viability, induced apoptosis and suppressed STAT3 and MYC signalling. Moreover, Rux synergized with bortezomib in myeloma cell lines and primary patient samples but spared normal cells. Collectively, these findings identify CD47 as an oncogenic driver with an intrinsic signalling function in MM and highlight the CD47-STAT3-MYC axis as a promising therapeutic target.
O. Faruq, Deepak Iyer, Patrick Wang et al.· British Journal of Haematolo...· 0 citations
