Journal: Blood Advances

Article Title: Rational polypharmacological targeting of FLT3, JAK2, ABL, and ERK1 suppresses the adaptive resistance to FLT3 inhibitors in AML

doi: 10.1182/bloodadvances.2022007486

Figure Lengend Snippet: Pluripotin potently inhibits the proliferation of Ba/F3 cells expressing FLT3 ITD , BCR-ABL, and Jak2 V617F . (A) The sigmoidal curve showing the viability of Ba/F3 cells expressing FLT3 ITD , BCR-ABL, and Jak2 V617F treated with dimethyl sulfoxide or increasing concentrations of pluripotin for 72 hours. IC 50 values for each cell line is indicated on the right. (B) Showing pFLT3, pERK1/2, and pSTAT5 levels determined by western blotting using total cells extracts incubated with different concentrations of pluripotin for 2 hours were probed with anti-pFLT3, anti-pSTAT5, anti-pERK1/2, anti-FLT3, anti-ERK1/2, and anti-STAT5 antibodies. (C) Scatter plot showing the extent of apoptosis in Ba/F3 cells expressing FLT3 ITD , BCR-ABL, and Jak2 V617F after the treatment with 100 nM of pluripotin. The quadrant in each plot show; Q1, early apoptotic cells (annexin V + ); Q2, late apoptotic cells (annexin V + and PI + ); Q3, necrotic cells (PI + ); and Q4, viable cells (annexin V − and PI − ). Representative cell proliferation data is shown from the mean of 2 independent experiments ± SD. Pluri, pluripotin.

Article Snippet: ABL inhibitors, imatinib, and nilotinib were obtained from LC Chemicals (Woburn, MA), whereas FLT3 inhibitors and MAPK pathway inhibitors were purchased from AdooQ Biosciences (Irvine, CA).

Techniques: Expressing, Western Blot, Incubation

Journal: Blood Advances

Article Title: Rational polypharmacological targeting of FLT3, JAK2, ABL, and ERK1 suppresses the adaptive resistance to FLT3 inhibitors in AML

doi: 10.1182/bloodadvances.2022007486

Figure Lengend Snippet: Pluripotin abolishes the adaptive resistance conferred by RAS mutants , BCR-ABL, and Jak2 V617 . Dose-response sigmoidal curve showing the proliferation of Ba/F3 cells expressing FLT3 ITD and cooperating mutants BCR-ABL, RAS G12V , and Jak2 V617F implicated in adaptive resistance. A dose-dependent cell proliferation showing resistance to gilteritinib (A) although treatment with pluripotin fully inhibited their proliferation (B). (C) A comparative immunoblot analysis showing inhibition of FLT3, ERK1/2, and STAT5. Total cell extracts from the BaF3-FLT3 ITD and BaF3-FLT3 ITD /RAS G12V cells treated with gilteritinib or pluripotin for 2 hours were probed with anti-pFLT3, anti-pSTAT5, anti-pERK1/2, anti-FLT3, anti-ERK1/2, and anti-STAT5 antibodies. The sigmoidal curves showing the proliferation of human AML cell lines, MOLM13 and MV4-11, expressing Ras G12V and RAS Q61K resistant to gilteritinib (D) whereas pluripotin treatment fully inhibited their proliferation (E). (F) Immunoblot analysis showing inhibition of proliferation is owing to on-target inhibition of FLT3, ERK1/2, and STAT5. Total cell extracts from the AML cells treated with pluripotin for 2 hours were probed with anti-pFLT3, anti-pSTAT5, anti-pERK1/2, anti-FLT3, anti-ERK1/2, and anti-STAT5 antibodies. Representative cell proliferation data and western blottings are shown from 2 independent experiments. Error bars represent ± SD.

Article Snippet: ABL inhibitors, imatinib, and nilotinib were obtained from LC Chemicals (Woburn, MA), whereas FLT3 inhibitors and MAPK pathway inhibitors were purchased from AdooQ Biosciences (Irvine, CA).

Techniques: Expressing, Western Blot, Inhibition

Journal: Blood Advances

Article Title: Rational polypharmacological targeting of FLT3, JAK2, ABL, and ERK1 suppresses the adaptive resistance to FLT3 inhibitors in AML

doi: 10.1182/bloodadvances.2022007486

Figure Lengend Snippet: Pluripotin is a type II inhibitor of FLT3, ABL, and Jak2 and a potent inhibitor of gatekeeper FLT3 mutant, F691L. Ribbon depiction of a structure of FLT3 with quizartinib (A) and with pluripotin (B). Ribbon depiction of Jak2-coordinate with type II inhibitor BBT594 (C) and with pluripotin (D). A ribbon cartoon of ABL-coordinate with type II inhibitor nilotinib (E) and with pluripotin (F). A stick representation of active sites of FLT3 (G), JAK2 (H), and ABL (I) showing pluripotin interaction with residues from the hinge region, catalytic glutamate of helix-C, and the conserved aspartate from the HRD motif. Overlapping binding of pluripotin is shown below. (J) Pluripotin binding is incompatible with active kinase conformation as phenylalanine residue of the DFG motif will block the binding (shown on the red surface) whereas inactive conformation is accessible when Phe will move out (shown on the green surface). (K) A close-up view of the active site of the FLT3 kinase showing the interaction of pluripotin with the gatekeeper, Phe 691 (top). A model showing that the gatekeeper mutation, F691L, will not affect pluripotin binding (bottom). (L) Sigmoidal curve showing that pluripotin is equally active on FLT3 ITD and its gatekeeper mutant, although mutants from the activation loop are resistant. Nonetheless, a significant therapeutic index (TI) exists between the activation loop mutants and normal Ba/F3 cells suggesting that dose escalation will suppress these variants. (M) Active site of Jak2 kinase showing the interaction of pluripotin with Leu 983 (top). A phenyl alanine substitution for Leu 983 will confer steric hindrance (bottom). (N) According to prediction, Jak2 V617F/L983F mutant conferred resistance to pluripotin. Thus, confirming the model and on-target inhibitory activity of pluripotin. (O) Active site of ABL kinase domain showing the interaction of pluripotin with the gatekeeper residue, Thr 315 (top). An isoleucine substitution for Thr 315 will confer steric hindrance (bottom). (P) As expected from the modeling studies, BCR-ABL T315 mutant conferred resistance to pluripotin. Together, these modeling and in vitro validation data confirm that pluripotin directly binds with inactive confirmations of FLT3, JAK2, and ABL.

Article Snippet: ABL inhibitors, imatinib, and nilotinib were obtained from LC Chemicals (Woburn, MA), whereas FLT3 inhibitors and MAPK pathway inhibitors were purchased from AdooQ Biosciences (Irvine, CA).

Techniques: Mutagenesis, Binding Assay, Blocking Assay, Activation Assay, Activity Assay, In Vitro

Journal: Blood Advances

Article Title: Rational polypharmacological targeting of FLT3, JAK2, ABL, and ERK1 suppresses the adaptive resistance to FLT3 inhibitors in AML

doi: 10.1182/bloodadvances.2022007486

Figure Lengend Snippet: Pluripotin effectively suppresses the TKI-resistant primary AML leukemia induced by FLT3 ITD /Tet2 -/- . (A) Experimental design of in vivo experiments to test the efficacy of pluripotin in FLT3 ITD / Tet2 -/- –induced AML using ROSACre ERT2 : FLT3 ITD : Tet2 fl/fl mice. (B) Survival curve showing the significant protection of mice with treatment with pluripotin compared with gilteritinib-treated cohort. (C-D) Shown are weekly progression of leukemic cells (CD45.2) and WBC from the PB of mice treated with gilteritinib and pluripotin. Note, pluripotin treatment effectively suppressed the leukemic progression compared with gilteritinib. Weekly blood counts, total red blood cells (RBCs) (E), hematocrit (HCT) (F), and platelets (G) did not show any adverse effect on hematopoiesis suggesting that pluripotin will be safe and effective. Presented data are from 2 independent experiments (5 mice per group) shown as mean ± SD. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001.

Article Snippet: ABL inhibitors, imatinib, and nilotinib were obtained from LC Chemicals (Woburn, MA), whereas FLT3 inhibitors and MAPK pathway inhibitors were purchased from AdooQ Biosciences (Irvine, CA).

Techniques: In Vivo

Journal: Blood Advances

Article Title: Rational polypharmacological targeting of FLT3, JAK2, ABL, and ERK1 suppresses the adaptive resistance to FLT3 inhibitors in AML

doi: 10.1182/bloodadvances.2022007486

Figure Lengend Snippet: Pluripotin treatment suppresses refractory/relapsed primary human leukemia. (A) Experimental design for the analysis of pluripotin treatment on AML cells from the refractory/relapsed patients. Shown is the survival of NSGS mice transplanted with primary human AML cells from AML1 (B) and AML4 (C). Patient AML1 has only FLT3 ITD mutation though AML4 harbors mutation in FLT3 ITD , RAS G13D , and PTPN11 D61V ( supplemental Figure 5 A). Note, pluripotin treatment with pluripotin significantly prolonged the survival, whereas gilteritinib was ineffective. Shown are leukemic burden measured by hCD45 levels in mice recipients of AML1 (D) and AML4 (E). Presented data are from 2 independent experiments (3 mice per group) shown as mean ± SD. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. ns, not significant.

Article Snippet: ABL inhibitors, imatinib, and nilotinib were obtained from LC Chemicals (Woburn, MA), whereas FLT3 inhibitors and MAPK pathway inhibitors were purchased from AdooQ Biosciences (Irvine, CA).

Techniques: Mutagenesis

Journal: Frontiers in Chemistry

Article Title: Urea-based anticancer agents. Exploring 100-years of research with an eye to the future

doi: 10.3389/fchem.2022.995351

Figure Lengend Snippet: Summary of cited patents.

Article Snippet: TW201706256A , Sunshine Lake Pharma , 2017 , FLT3 Inhibitors , Preclinical phase.

Techniques: