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. 2019 Nov 26;3(22):3661-3673.
doi: 10.1182/bloodadvances.2019001068.

LAM-003, a new drug for treatment of tyrosine kinase inhibitor-resistant FLT3-ITD-positive AML

Neil Beeharry  1 , Sean Landrette  1 , Sophia Gayle  1 , Marylens Hernandez  1 , Jeff E Grotzke  1 , Peter R Young  1 , Paul Beckett  1 , Xuan Zhang  2 , Bing Z Carter  2 , Michael Andreeff  3 , Stephanie Halene  4 , Tian Xu  5 , Jonathan Rothberg  1 , Henri Lichenstein  1
Affiliations

LAM-003, a new drug for treatment of tyrosine kinase inhibitor-resistant FLT3-ITD-positive AML

Neil Beeharry et al. Blood Adv. .

Abstract

Acute myeloid leukemias (AML) harboring a constitutively active internal tandem duplication (ITD) mutation in the FMS-like kinase tyrosine kinase (FLT3) receptor are associated with poor patient prognosis. Despite initial clinical responses to FLT3 kinase inhibitors, patients eventually relapse. Mechanisms of resistance include the acquisition of secondary FLT3 mutations and protective stromal signaling within the bone marrow niche. Here we show that LAM-003, a prodrug of the heat shock protein 90 inhibitor LAM-003A, has cytotoxic activity against AML cell lines and primary samples harboring FLT3-ITD. LAM-003 regressed tumors in an MV-4-11 xenograft mouse model and extended survival in a MOLM-13 systemic model. LAM-003 displayed synergistic activity with chemotherapeutic drugs and FLT3 inhibitors, with the most robust synergy being obtained with venetoclax, a BCL-2 inhibitor. This finding was verified in a MOLM-13 systemic survival model in which the combination significantly prolonged survival compared with the single agents. Importantly, LAM-003 exhibited equipotent activity against FLT3 inhibitor-resistant mutants of FLT3, such as D835 or F691, in cytotoxic and FLT3 degradation assays. LAM-003 also retained potency in AML cells grown in stromal-conditioned media that were resistant to FLT3 inhibitors. Lastly, a genome-wide CRISPR screen revealed epigenetic regulators, including KDM6A, as determinants of LAM-003 sensitivity in AML cell lines, leading to the discovery of synergy with an EZH2 inhibitor. Collectively, these preclinical findings support the use of LAM-003 in FLT3-ITD patients with AML who no longer respond to FLT3 inhibitor therapy either as a single agent or in combination with drugs known to be active in AML.

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Conflict of interest statement

Conflict-of-interest disclosure: N.B., S.L., S.G., J.E.G., P.B., M.H., P.R.Y., and H.L. are employees at AI Therapeutics. T.X. is on the AI Therapeutics advisory board. J.R. is a Director of AI Therapeutics. AI Therapeutics is the owner of LAM-003/LAM-003A patents. The remaining authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
LAM-003 display antileukemic activity in AML cells harboring FLT3-ITD and synergistic activity with venetoclax. (A) Dot plot of average EC50 values in AML cell lines harboring WT FLT3 (WT) (n = 12) or FLT3-ITD (n = 3) or primary blasts (FLT3 WT, n = 7; FLT3-ITD and/or D835 mutation, n = 18) treated with LAM-003 for 72 hours. Cell lines were tested in duplicate a minimum of 2 independent times, and primary samples were tested once, in duplicate. The geometric mean ± 95% confidence interval is shown. Open circles are primary samples harboring D835 mutations, half circles are primary samples harboring FLT3-ITD and D835 mutation. EC50 values were calculated as described in the supplemental Methods and materials and shown in supplemental Table 1. *P < .05 using unpaired Student t test with Welch’s correction. (B) Expression of FLT3 and phospho-S6, phospho-AKT, phospho-STAT5, and phospho-SYK/ZAP-70 was evaluated by using flow cytometry in MV-4-11 (green bars) and MOLM-13 (pink bars) cells after treatment with LAM-003 for 24 hours. Average data ± standard deviation (SD) from 2 independent experiments are shown. (C) Normalized isobologram at the EC75 of three FLT3-ITD–harboring cell lines treated with a combination of LAM-003 and venetoclax for 72 hours before viability was assayed by using CellTiter-Glo. Each data point is the average of 2 independent experiments, each performed in duplicate. (D) Western blot analysis of MOLM-14 cells treated with LAM-003 (1 µM), venetoclax (20 nM), or the combination for 24 hours. Lysates were probed with antibodies to PARP or vinculin, which was used as a loading control. Upper and lower arrows denote full-length PARP and cleaved PARP, respectively. Representative data shown from 2 independent experiments. (E) EC50 values of LAM-003 or venetoclax in parental MOLM-13 or MV-4-11 cells and venetoclax-resistant cell lines (Ven-R) treated for 72 hours before viability determined by using CellTiter-Glo luminescent cell viability reagent. Experiments were performed a minimum of 2 independent times, each in duplicate, and averaged data ± SD are shown. (F) Western blot analysis of MOLM-14 cells treated with LAM-003 (1 µM), venetoclax (20 nM), or the combination for 24 hours. Lysates were probed with the indicated antibodies. Vinculin was used as a loading control. Representative blot shown from 2 independent experiments.
Figure 2.
Figure 2.
LAM-003 exhibits potent antileukemic activity in AML mouse models. (A) Kaplan-Meier survival analysis of a MOLM-13 systemic model in which mice were dosed orally with vehicle or with LAM-003 (75 or 150 mg/kg daily). Statistical significance was calculated by using the log-rank (Mantel-Cox) test. **P = .005 for LAM-003 75 mg/kg vs vehicle; **P = .008 for LAM-003 150 mg/kg vs vehicle. (B) Kaplan-Meier analysis of animal survival of mice inoculated with MOLM-13 cells systemically and treated with LAM-003 orally, venetoclax orally, or the combination as indicated once daily. Statistical significance was calculated by using the log-rank (Mantel-Cox) test. **P = .008 for LAM-003 vs vehicle; ***P = .0006 for the drug combination vs vehicle.
Figure 3.
Figure 3.
LAM-003 overcomes mutations that confer FLT3i resistance. (A) Ba/F3 cells harboring WT FLT or indicated FLT3 mutants were treated with increasing concentrations of LAM-003 for 24 hours before flow cytometry was performed to assess FLT3 expression. (B) Ba/F3 cells expressing FLT3-ITD or FLT3-ITD F691L were treated with crenolanib or LAM-003 for 72 hours. (C) Parental MOLM-13 cells (solid lines) or MOLM13-RES cells (hatched lines) were treated with tandutinib, sorafenib, or LAM-003 at the indicated concentrations for 72 hours before viability was assayed by using CellTiter-Glo. Data in all panels represent the mean ± SD from at least 2 independent experiments, each performed in duplicate.
Figure 4.
Figure 4.
LAM-003 overcomes intrinsic FLT3 TKI resistance mechanisms. (A) AML cell lines were treated with LAM-003, gilteritinib, or crenolanib for 72 hours in either conditioned medium or nonconditioned medium before cell viability was determined by using CTG. All comparisons were made to LAM-003 by using a 1-way analysis of variance, Dunnett’s multiple comparisons test. For MOLM-13, **P = .007, ****P < .0001; for MOLM-14, ***P = .0002, ****P < .0001; for MV-4-11, **P = .002, ****P < .0001. (B) AML cell lines were treated with LAM-003 or venetoclax for 72 hours in either conditioned medium or nonconditioned medium. For MOLM-13, **P = .001; for MOLM-14, **P < .005; for MV-4-11, **P < .007 compared with LAM-003 using 2-way analysis of variance, Bonferroni’s multiple comparisons test. (C) Normalized isobologram at the EC75 of MV-4-11 cells and MOLM-13 cells with the combination of LAM-003 and venetoclax in nonconditioned (solid symbol) or conditioned (open symbol) medium for 72 hours before viability was assayed by using CellTiter-Glo. Each data point is the average of 2 independent experiments, each performed in duplicate, for each cell line. (D) EC50 values of BA/F3 cells expressing WT-FLT3 or FLT3-ITD supplemented with or without IL-3 and treated with gilteritinib, crenolanib, or LAM-003. Viability was assessed 72 hours later by using CTG. Data in all panels are the mean of at least 2 independent experiments, each performed in duplicate. Bar graphs in panels A-B and D represent the mean ± SD.
Figure 5.
Figure 5.
LAM-003 activity against TKI-resistant primary AML blasts harboring FLT3-ITD. (A) Three primary AML samples all harboring FLT3-ITD were treated with LAM-003 for 72 hours before Annexin V/7-AAD staining was assessed on CD45+/CD34+ blasts. (B) Primary AML blasts from a TKI-relapsed patient were treated with gilteritinib (left) or LAM-003 (right) for 72 hours before cell viability was assessed by using 7-AAD/Annexin V staining as in panel A. (C) Normalized isobologram at the EC75 of 2 primary AML samples, both harboring FLT3-ITD, with the combination of LAM-003 and venetoclax for 72 hours before being assayed for Annexin V/7-AAD staining as in panel A.
Figure 6.
Figure 6.
CRISPR identifies epigenetic regulation as a key determinant of LAM-003 sensitivity. (A) Scatter plot showing enrichment of the top 20 normalized sgRNA read counts in vehicle and LAM-003–treated CRISPR pools in GeCKO sublibraries A and B. (B) Gene ontology analysis of top 20 sgRNAs. (C) Scatter plot showing enrichment of normalized sgRNA read count of KDM6A in vehicle and LAM-003–treated CRISPR pools from the combined A and B GeCKO sublibraries. Six individual sgRNAs used for targeting KDM6A are shown in red. (D) Normalized isobologram at the EC75 of two FLT3-ITD–harboring cell lines treated with the EZH2 inhibitor EPZ6438 for 4 days followed by the combination of EZH2 inhibitors and LAM-003 for an additional 72 hours before viability was assayed by using CellTiter-Glo. Data points are the average of 2 independent experiments, each performed in duplicate.
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