Introduction: In acute myeloid leukemia (AML) standard therapies induce complete remission in 50-70% of patients, but overall two-year survival is less than 20-30% because of high relapse rates. AML with TP53 alteration is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplantation. Mutations in TP53 are seen in approximately 20% of AML and confer a particularly poor prognosis decreasing the 1-2 year survival rates to 0-10%. Leukemia stem cells (LSCs) are defined as the cells that recapitulate the leukemia and cause relapse. Recent large-scale drug-screening efforts to map treatment vulnerabilities in AML have revealed that crizotinib can reduce the in vitro viability of unselected primary human AML bone marrow samples with TP53 alterations (Tyner et al. Nature 2018). Crizotinib is a tyrosine kinase inhibitor that targets ALK, MET, and RON and is FDA approved for treatment of lung cancer. Since LSCs are largely responsible for AML mortality, we investigated whether crizotinib targets LSCs in primary, diagnostic human AML samples with TP53 alterations. We used mass cytometry (CyTOF2) to determine the cell-type specific effects of this drug on intracellular signaling states. We profiled treated samples with a panel that includes immunophenotypic markers (including LSC markers) and intracellular signaling molecules implicated in AML pathogenesis and aggressiveness. Results: Primary human AML samples with TP53 alterations were treated with crizotinib (10 mM, n=3). There was a reduction of viability by 48 hours of in vitro treatment with crizotinib, relative to vehicle (mean 64% reduction in viability). Crizotinib abolished all colony formation in semi-solid media. Mass cytometry analysis revealed that crizotinib treatment led to a loss of cells bearing LSC-specific markers such as CD123 and TIM3. Crizotinib treatment also induced a consistent loss of phosphorylated-signaling intermediates of the STAT and p38/MAPAPKII pathways in all samples tested. Notably, crizotinib treatment also induced significant reductions in total and phosphorylated NFKB in all samples tested. Conclusions: These data reveal cell-type specific effects of crizotinib in human AML with TP53 alterations. Crizotinib induced a specific loss of LSCs and inhibition of the STAT, p38/MAPAPKII and NFKB pathways. These data suggest that crizotinib inhibition may target LSCs in AML with TP53 alterations. Figure Disclosures No relevant conflicts of interest to declare.
In acute myeloid leukemia (AML), the impact of genetic drivers on response to therapy and long-term survival has been well characterized. AML with complex cytogenetics and TP53 alterations (TP53Alt) is a poor-risk AML subtype that is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplant leading to survival rates 0-10% at 1 year. In contrast, AML with favorable risk molecular features is highly sensitive to chemotherapy and confers survival rates of 50-70%. AML with intermediate risk molecular features can be responsive to chemotherapy and can be cured with hematopoietic stem cell transplant leading to overall survival rates of 30-60%. Leukemia stem cells (LSCs), the cells that recapitulate and propagate leukemia, are central to leukemia progression and relapse. Given the differences in chemo-sensitivity and clinical behavior of genetic subgroups of AML, we asked whether LSCs from poor risk AMLs exhibit distinct signaling activation profiles. We assembled a panel of 23 primary human AML samples with intermediate- and poor- risk genetics and used CyTOF (mass cytometry) to quantitatively measure the levels of immunophenotypic proteins and intracellular signaling molecules in each sample, at the single-cell level. We gated on CD34+CD123+CD3-CD19- cells (LSCs) and measured the level of intracellular signaling molecules within the LSCs of each sample. Notably, the intracellular signaling activation state of LSCs from each AML subtype was distinct; NFkB, pERK, p4EBP1, and pSTAT3 were uniquely upregulated in complex cytogenetics and TP53Alt LSCs, relative to LSCs from intermediate risk AML, suggesting that these signaling pathways may be important for LSC function in this AML subtype. Given that TP53Alt independently confer treatment resistance in AML, we focused on this genetic subgroup. We compared the gene expression profiles of TP53Alt and TP53-wild-type AML samples from the BEAT AML dataset (Tyner et al. Nature 2018) and found that the gene expression profiles of TP53Alt samples are enriched for gene sets representing JAK/STAT signaling, consistent with our CyTOF data, which identified activation of STAT3 in TP53Alt LSCs. A recent drug screen in AML demonstrated that a JAK1/2 kinase inhibitor, AZD1480, can reduce the in vitro viability of TP53-deleted AML cell lines (Nechiporuk et al. Ca Discovery 2019), but these effects were not tested in primary AML samples or on LSCs. Since LSCs confer treatment resistance, we investigated the effect of the AZD1480 on the LSC population in TP53Alt primary human AML samples. AZD1480 treatment abolished all colony formation in primary human TP53Alt AML samples (n=7, 6 replicates per sample, p<0.01). Treatment of these samples in liquid cultures led to a 50% reduction in LSC frequency. We used CyTOF to profile the intracellular signaling states of in vitro treated samples and found that AZD1480 attenuated pSTAT3, pSTAT5, p4EBP1, and NFkB in the LSCs of these samples. The mTOR/4EBp1 and NF༆B pathways have been implicated as drivers of self-renewal and LSC function in AML. Our data suggest that JAK/STAT inhibition may target these pathways in TP53Alt LSCs. These data demonstrate the unique signaling states of TP53Alt LSCs, relative to other LSCs, and show that inhibition of the JAK/STAT pathway specifically targets LSCs within human TP53Alt AML. Figure Disclosures No relevant conflicts of interest to declare.
Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow with a low two‐year survival rate. Standard chemotherapy achieves complete remission in 60–80% of patients, but 20–30% patients relapse due to leukemia stem cells (LSCs) that can self‐renew and recapitulate disease. AML with TP53 alterations has a lower prognosis with survival rates of 0–10% at one year. In vitro drug screens have identified drugs to target AML, but none have studied the effect of these therapies on LSCs with TP53 alterations. These in vitro drug screens include Crizotinib, Elesclomol, AZD1480, GW2580, Venetoclax, and Entrectinib. Crizotinib, AZD1480, GW2580, Venetoclax, and Entrectinib are signaling pathway inhibitors. Elesclomol induces oxidative stress and apoptosis in cancer cells. The goal of this research is to understand the molecular mechanisms of self‐renewal and therapeutic vulnerabilities in LSCs of AML with TP53 alterations. Specifically, we investigate whether these agents target LSCs from AML with TP53 alterations. We performed in vitro viability and in vitro colony forming assays (CFAs) on primary human AML samples with TP53 alterations, plating them with drugs or vehicle control. Furthermore, we used CYTOF to assess the effects of each drug on signaling within leukemia subpopulations. Crizotinib proved to be effective in reducing in vitro viability, in vitro CFAs, and affecting the expression of immunophenotypic markers, such as CD34 and CD123, which are markers for leukemia stem cell populations. Our data reveals cell‐type specific effects of Crizotinib in human AML with TP53 alterations. Our findings suggest that Crizotinib may be an effective therapy for patients with AML with TP53 alterations. Support or Funding Information This research was supported and funded by: NIH/NIGMS MARC U* STAR T34 HHS 00026 National Research Service Award to UMBC, Summer Research at the University of Minnesota Medical School NIH HLBI 2R25HL088728‐11A1, Dr. Colin Campbell, Dr. Craig Henke, American Cancer Society Mentored Research Scholar Grant (MSRG‐16‐195‐01‐DDC), Frederick A. Deluca Foundation, CTSI K to R01 Award, NIH/NCATS, Lois and Richard King Assistant Professorship in Medicine, Division of Hematology, Oncology, Transplantation, Dept. of Medicine, University of Minnesota University of Minnesota Foundation Donors
Mathematical models formally and precisely represent biological mechanisms with complex dynamics. To understand the possible behaviors of such systems, phase portrait diagrams can be used to visualize their overall global dynamics across a domain. However, producing these phase portrait diagrams is a laborious process reserved to mathematical experts. Here, we developed a computational methodology to automatically generate phase portrait diagrams to study biological dynamical systems based on ordinary differential equations. The method only needs as input the variables and equations describing a multidimensional biological system and it automatically outputs for each pair of dependent variables a complete phase portrait diagram, including the critical points and their stability, the nullclines of the system, and a vector space of the trajectories. Crucially, the portraits generated are interactive, and the user can move the visualized planar slice, change parameters with sliders, and add trajectories in the phase and time domains, after which the diagrams are updated in real time. The method is available as a user-friendly graphical interface or can be accessed programmatically with a Mathematica package. The generated portraits and particular views can be saved as computable notebooks preserving the interactive functionality, an approach that can be adopted for reproducible science and interactive pedagogical materials. The method, code, and examples are freely-available at https://lobolab.umbc.edu/autoportrait .
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