Squamous cell carcinoma (SCC) of the lung is the second most common subtype of lung cancer. With limited treatment options, the 5-year survival rate of SCC is only 15%. Although genomic alterations ...in SCC have been characterized, identifying the alterations that drive SCC is critical for improving treatment strategies. Mouse models of SCC are currently limited. Using lentiviral delivery of Sox2 specifically to the mouse lung, we tested the ability of Sox2 to promote tumorigenesis in multiple tumor suppressor backgrounds. Expression of Sox2, frequently amplified in human SCC, specifically cooperates with loss of Lkb1 to promote squamous lung tumors. Mouse tumors exhibit characteristic histopathology and biomarker expression similar to human SCC. They also mimic human SCCs by activation of therapeutically relevant pathways including STAT and mTOR. This model may be utilized to test the contribution of additional driver alterations in SCC, as well as for preclinical drug discovery.
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•Lentiviral approach identifies combinatorial gene drivers in lung cancer•Sox2 expression and Lkb1 loss cooperate to promote lung squamous cell carcinoma•Mouse lung tumors recapitulate human SCC histology and biomarker expression•SCCs display activation of potential therapeutic targets FGFR2, STAT3, NF-κB, and mTOR
Sox2 is one of the most frequently altered genes in squamous cell lung cancer, but its role in lung tumorigenesis is unclear. Using a lentiviral approach, Mukhopadhyay et al. show that Sox2 expression in the mouse lung cooperates specifically with loss of the tumor suppressor Lkb1 to promote squamous lung tumors. Mouse lung tumors recapitulate human squamous tumors in histopathology and biomarker expression and exhibit activation of potential targets for therapy.
Tumor necrosis factor alpha (TNF) is increased in myelofibrosis (MF) and promotes survival of malignant over normal cells. The mechanisms altering TNF responsiveness in MF cells are unknown. We show ...that the proportion of marrow (BM) cells expressing TNF is increased in MF compared to controls, with the largest differential in primitive cells. Blockade of TNF receptor 2 (TNFR2), but not TNFR1, selectively inhibited colony formation by MF CD34
and mouse JAK2
progenitor cells. Microarray of mouse MPN revealed reduced expression of X-linked inhibitor of apoptosis (Xiap) and mitogen-activated protein kinase 8 (Mapk8) in JAK2
relative to JAK2
cells, which were normalized by TNFR2 but not TNFR1 blockade. XIAP and MAPK8 were also reduced in MF CD34
cells compared to normal BM, and their ectopic expression induced apoptosis. Unlike XIAP, expression of cellular IAP (cIAP) protein was increased in MF CD34
cells. Consistent with cIAP's role in NF-κB activation, TNF-induced NF-κB activity was higher in MF vs. normal BM CD34
cells. This suggests that JAK2
reprograms TNF response toward survival by downregulating XIAP and MAPK8 through TNFR2. Our results reveal an unexpected pro-apoptotic role for XIAP in MF and identify TNFR2 as a key mediator of TNF-induced clonal expansion.
We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in ...regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.
•K0706 is a BCR-ABL1 tyrosine kinase inhibitor under clinical investigation in CML.•K0706 exhibits in vitro activity against most clinically important BCR-ABL1 point mutants.•Cell proliferation ...assays demonstrate limited activity of K0706 against BCR-ABLT315I.•Minor structural differences account for the differential activity of ponatinib and K0706 against BCR-ABLT315I.•K0706 appears to have a favorable toxicity profile based on early patient data.
BCR-ABL1 tyrosine kinase inhibitors (TKIs) are the cornerstone of treatment in chronic myeloid leukemia. Although there are now four TKIs approved for use in the front-line setting, acquired TKI resistance via secondary kinase domain mutations remains a problem for patients. K0706 is a novel BCR-ABL1 TKI currently under clinical investigation with structural elements similar to those of ponatinib and dasatinib. In this article, we functionally characterize the anti-leukemic activity of K0706 using cell proliferation assays in conjunction with drug resistance screening. We provide details from molecular modeling to support our in vitro findings and additionally describe our limited clinical experience with this drug in two patients treated on trial. We demonstrate that although K0706 retains efficacy against a large spectrum of clinically relevant mutations, it does not appear to have activity against BCR-ABL1T315I. Early trial experience suggests excellent tolerability, which may positively affect the place of K0706 within the ever-expanding chronic myeloid leukemia treatment paradigm.
The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and ...differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common β-chain (βc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo.
•Low MS4A3 is a common mechanism among LSPC quiescence, BCR-ABL1-independent primary TKI resistance, and blastic transformation.•MS4A3 controls LSPC sensitivity to differentiating cytokines by regulating βc receptor endocytosis and signaling.
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Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive ...activation of JAK/STAT signaling through mutations in
, or
is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis.
A short hairpin RNA library screening was performed on JAK2
-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including
and
were among top candidates. JAK2
-mutant cell lines, human primary myelofibrosis CD34
cells, and a retroviral JAK2
-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor).
JAK2
-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34
cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34
cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2
-mutant cells
.
Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.
Drug-free macromolecular therapeutics (DFMT) is a new paradigm for the treatment of B cell malignancies. Apoptosis is initiated by the biorecognition of complementary oligonucleotide motifs at the ...cell surface resulting in crosslinking of CD20 receptors. DMFT is composed from two nanoconjugates: 1) bispecific engager, Fab’-MORF1 (anti-CD20 Fab’ fragment conjugated with morpholino oligonucleotide), and 2) a crosslinking (effector) component P-(MORF2)X (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple copies of complementary morpholino oligonucleotide). We evaluated this concept in 44 samples isolated from patients diagnosed with various subtypes of B cell malignancies. Apoptosis was observed in 65.9% of the samples tested. Pretreatment of cells with gemcitabine (GEM) or polymer-gemcitabine conjugate (2P-GEM) enhanced CD20 expression levels thus increasing apoptosis induced by DFMT. These positive results demonstrated that DFMT has remarkable therapeutic potential in various subtypes of B cell malignancies.
Drug-free macromolecular therapeutics (DFMT) is a new paradigm for the treatment of B cell malignancies. Apoptosis is initiated by crosslinking of CD20 receptors triggered by the biorecognition of complementary oligonucleotide motifs at the cell surface. The therapeutic efficacy of DFMT on 44 primary patient samples with various B-cell malignancies has been evaluated. Pretreatment of cells with gemcitabine or polymer-gemcitabine conjugate enhanced CD20 expression and increased levels of apoptosis. The results indicate that DFMT has significant translational potential. Display omitted
Highlights•• K0706 is BCR-ABL1 tyrosine kinase inhibitor under clinical investigation in CML. •• K0706 exhibits in vitro activity against most clinically important BCR-ABL1 point mutants. •• Cell ...proliferation assays demonstrate limited activity of K0706 against BCR-ABLT315I. •• Minor structural differences account for the differential activity of ponatinib and K0706 against BCR-ABLT315I. •• K0706 appears to have a favorable toxicity profile based on early patient data.
Normal human bone marrow cells are critical for studies of hematopoiesis and as controls to assess toxicity. As cells from commercial vendors are expensive, many laboratories resort to cancer-free ...bone marrow specimens obtained during staging or to umbilical cord blood cells, which may be abnormal or reflect a much younger age group compared to the disease samples under study. We piloted the use of femoral heads as an alternative and inexpensive source of normal bone marrow. Femoral heads were obtained from 21 successive patients undergoing elective hip arthroplasty. Mononuclear cells (MNCs) were purified with Ficoll, and CD3+, CD14+, and CD34+ cells were purified with antibody-coated microbeads. The median yield of MNCs was 8.95 × 107 (range, 1.62 × 105-2.52 × 108), and the median yield of CD34+ cells was 1.40 × 106 (range, 3.60 × 105-9.90 × 106). Results of downstream applications including qRT-PCR, colony-forming assays, and ex vivo proliferation analysis were of high quality and comparable to those obtained with standard bone marrow aspirates. We conclude that femoral heads currently discarded as medical waste are a cost-efficient source of bone marrow cells for research use.
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