Pim Kinase Inhibitors Increase Gilteritinib Cytotoxicity in FLT3-ITD Acute Myeloid Leukemia Through GSK-3β Activation and c-Myc and Mcl-1 Proteasomal Degradation

Abstract Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) has poor outcomes. FLT3-ITD drives constitutive and aberrant FLT3 signaling, activating STAT5 and upregulating the downstream oncogenic serine/threonine kinase Pim-1. FLT3 inhibitors are in clinical use, but with limited and transient efficacy. We previously showed that concurrent treatment with Pim and FLT3 inhibitors increases apoptosis induction in FLT3-ITD–expressing cells through posttranslational downregulation of Mcl-1. Here we further elucidate the mechanism of action of this dual targeting strategy. Cytotoxicity, apoptosis and protein expression and turnover were measured in FLT3-ITD–expressing cell lines and AML patient blasts treated with the FLT3 inhibitor gilteritinib and/or the Pim inhibitors AZD1208 or TP-3654. Pim inhibitor and gilteritinib cotreatment increased apoptosis induction, produced synergistic cytotoxicity, downregulated c-Myc protein expression, earlier than Mcl-1, increased turnover of both proteins, which was rescued by proteasome inhibition, and increased efficacy and prolonged survival in an in vivo model. Gilteritinib and Pim inhibitor cotreatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids, preventing phosphorylation at these sites, did not downregulate these proteins, increase their turnover or increase apoptosis induction. Moreover, concurrent treatment with gilteritinib and Pim inhibitors dephosphorylated (activated) the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β), and GSK-3β inhibition prevented c-Myc and Mcl-1 downregulation and decreased apoptosis induction. The data are consistent with c-Myc T58 and Mcl-1 S159 phosphorylation by activated GSK-3β as the mechanism of action of gilteritinib and Pim inhibitor combination treatment, further supporting GSK-3β activation as a therapeutic strategy in FLT3-ITD AML. Significance: FLT3-ITD is present in 25% of in AML, with continued poor outcomes. Combining Pim kinase inhibitors with the FDA-approved FLT3 inhibitor gilteritinib increases cytotoxicity in vitro and in vivo through activation of GSK-3β, which phosphorylates and posttranslationally downregulates c-Myc and Mcl-1. The data support efficacy of GSK-3β activation in FLT3-ITD AML, and also support development of a clinical trial combining the Pim inhibitor TP-3654 with gilteritinib.


Introduction
Internal tandem duplication of the fms-like tyrosine kinase 3 receptor tyrosine kinase (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients (1).These patients respond to chemotherapy but relapse rapidly and have poor outcomes (2).While outcomes have improved with incorporation of FLT3 inhibitors and allogeneic hematopoietic stem cell transplantation into activates signal transducer and activator of transcription (STAT) 5, upregulating the downstream oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus 1 (Pim-1; refs.6, 7), the c-Myc oncogene (7) and the antiapoptotic protein Mcl-1 (8).Pim-1, one of three Pim kinase isoforms, Pim-1, Pim-2, and Pim-3, not only contributes directly to the proliferative and antiapoptotic effects of FLT3-ITD, but also phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD (9,10).Dual inhibition of Pim and FLT3 kinases has been shown to enhance cytotoxicity and apoptosis induction in cell lines and primary AML cells with FLT3-ITD (9)(10)(11)(12)(13)(14)(15).Pim kinase inhibitors are in clinical development (16,17) and combining Pim and FLT3 inhibitors is a promising treatment strategy for AML with FLT3-ITD.c-Myc is a transcription factor that dimerizes with its coactivator, Max, to induce expression of a number of gene families, driving both proliferation and resistance to apoptosis induction (18).In addition to being transcriptionally upregulated in AML with FLT3-ITD (7), c-Myc is also regulated posttranslationally by Pim-1 (19) and by the serine/threonine kinase glycogen synthase kinase-3β ref. 20).Pim-1-mediated phosphorylation of c-Myc at S62 and decreased phosphorylation at T58 result in posttranslational upregulation via stabilization (19), while GSK-3β phosphorylation of c-Myc at T58 promotes its proteasomal degradation (20).GSK-3β, in turn, is a substrate of both AKT (21) and Pim-1 kinase (22), both of which phosphorylate it at S9, thereby rendering it catalytically inactive (21,23).In addition, we showed that GSK-3β also phosphorylates Pim-1, resulting in its posttranslational downregulation, creating a negative feedback loop between these two kinases (24).
We previously showed that concurrent Pim and FLT3 inhibition increases apoptosis induction in cells with FLT3-ITD, but not wild-type FLT3, through posttranslational downregulation of the antiapoptotic protein Mcl-1 (12), which is also upregulated in AML with FLT3-ITD (8).Mcl-1 is also phosphorylated by GSK-3β, at S159, and thereby tagged for degradation (25).Here we demonstrate that Pim and FLT3 inhibitor cotreatment activates GSK-3β in cells with FLT3-ITD, resulting in posttranslational downregulation of both c-Myc and Mcl-1.

Patient Samples
Blood samples were obtained from patients with AML with FLT3-ITD with peripheral blasts on a University of Maryland School of Medicine Institutional Review Board-approved tissue procurement protocol, following written informed consent.Studies were conducted in accordance with the Declaration of Helsinki.Peripheral blood mononuclear cells were isolated by density centrifugation over Ficoll-Paque (Millipore Sigma, #GE17-1440-02).Patient sample clinical information is summarized in Supplementary Table S1.

Drug Combination Studies
Cells were seeded in a 96-well plate and treated in triplicate with gilteritinib and Pim inhibitor at various concentrations alone and in combinations.WST-1 Cell Proliferation Reagent (Millipore Sigma, #11644807001) was added after 48 hours to terminate the assay.Drug combination effects were determined using the Chou-Talalay method, analyzed with CompuSyn software (Com-boSyn; RRID:SCR 022931; ref. 34).
In a first experiment, treatment was initiated with gilteritinib 7.5 mg/kg and/or TP-3654 50 mg/kg in 5% DMSO, 40% polyethylene glycol 300 (PEG 300), 5% polysorbate 80 (Tween 80) and 50% water, or vehicle control, all by oral gavage, once every other day for three doses, followed by 2 days rest, each week.Gilteritinib dose and schedule were chosen to enhance demonstration of synergy.
In a second experiment, mice were treated with gilteritinib 15 mg/kg and/or TP-3654 50 mg/kg, or vehicle control, 5 days per week each week.Gilteritinib administered at 10 mg/kg orally daily was previously shown to effectively suppress FLT3 and STAT5 phosphorylation in an MV4-11 mouse model, leading to sustained antitumor activity (35).Gilteritinib reaches its maximum efficacy in patients at 80 mg or orally per day (29), with an approved clinical dose of 120 mg orally daily.Gilteritinib 15 mg/kg has been used previously in combination experiments in vivo (36).
Mice were weighed prior to each treatment.Leukemia burden was assessed weekly by noninvasive luciferin imaging, as above.Bioluminescent image data were analyzed with Living Image software (Revvity, #128113).Endpoints were 20% body weight loss, hind limb paralysis or lack of mobility to eat/drink.The University of Maryland Institutional Animal Care and Use Committee approved the study.

Protein Turnover and Proteasomal Degradation
To study protein turnover, cells were treated with 100 μg/mL cycloheximide (CHX; Millipore Sigma, #C7698) for 60 minutes to block new protein translation before adding gilteritinib and/or Pim inhibitor, or DMSO control.To study the effect of proteasomal degradation, cells were treated with CHX as above, with or without addition of the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132;Millipore Sigma, #474790; 20 μmol/L) 30 minutes after adding CHX and 30 minutes before gilteritinib and/or Pim inhibitor, or DMSO control, treatment.Protein expression was measured at serial timepoints by immunoblotting.Band intensities were measured by densitometry, as above, and 50% protein turnover timepoints were determined using the line of best fit.

Statistical Analysis
Flow cytometric analysis was performed on data from at least three independent experiments.Datapoints were pooled, with error bars representing SEM.Statistical analysis was performed by unpaired t test, using GraphPad Prism 9 (GraphPad Prism, GraphPad Software, RRID:SCR_002798).In the in vivo model, photon intensity in mice treated with TP-3654 and gilteritinib combination versus gilteritinib alone was compared by two-way ANOVA with Sidak multiple comparison test, and survival was compared by Kaplan-Meier analysis.Immunoblotting experiments were performed at least twice.
Single immunoblots and corresponding densitometric analysis are shown in figures.

Data Availability
The data generated in this study are available within the article.

FLT3 and Pim Inhibitor Cotreatment is Synergistic in Cells with FLT3-ITD
We previously showed that cotreatment with the pan-Pim inhibitor AZD1208 and the FLT3 inhibitor quizartinib enhanced cytotoxicity, compared with single drugs, in cells with FLT3-ITD, but not wild-type FLT3, in vitro and in vivo (12).Here we studied efficacy of the FDA-approved FLT3 inhibitor gilteritinib and AZD1208 or the novel pan-Pim-1 inhibitor TP-3654, currently in clinical trials (refs.37, 38; Fig. 1).Ba/F3-ITD, MV4-11 and MOLM-14 cells were treated with gilteritinib and/or AZD1208 (Fig. 1A; Supplementary Fig. S1) or TP-S3654 (Fig. 1C; Supplementary Fig. S1), or DMSO control, and apoptosis was measured by Annexin V/PI staining.Both combinations significantly increased apoptosis induction, compared with gilteritinib alone, in all three cell lines.Ba/F3-ITD, MV4-11, and MOLM-14 cells were also treated with gilteritinib and AZD1208 (Fig. 1B) or TP-3654 (Fig. 1D) as single drugs and combinations at different concentrations.Synergistic effects of both drug combinations were demonstrated by Chou-Talalay analysis in all three cell lines.

In Vivo Efficacy of TP-3654 and Gilteritinib
In the in vivo model (Fig. 2), luminescence decreased significantly over time in mice engrafted with MV4-11-luc cells treated with TP-3654 and gilteritinib, compared with gilteritinib alone (Fig. 2A, B, D, and E), and treatment with TP-3654 and gilteritinib combination significantly prolonged survival (Fig. 2C).

Pim and FLT3 Inhibitor Cotreatment Downregulates c-Myc and Mcl-1 Protein Posttranslationally, Through Increased Proteasomal Degradation
To determine the mechanism(s) by which gilteritinib and Pim inhibitor cotreatment downregulates c-Myc, Ba/F3-ITD, MV4-11, and MOLM-14 cells were pretreated for 1 hour with CHX with or without addition of MG-132 after 30 minutes, then treated with gilteritinib and/or AZD1208, or DMSO control (Fig. 4).c-Myc protein was measured by immunoblotting at serial timepoints, with vinculin loading control (Fig. 4A).AZD1208 and gilteritinib cotreatment accelerated c-Myc protein turnover, relative to single drugs or DMSO control, in all three cell lines, and accelerated turnover was abrogated by MG-132 pretreatment (Fig. 4C).Similarly, Mcl-1 protein was measured by immunoblotting at serial timepoints, with vinculin loading control (Fig. 4B).Mcl-1 protein turnover was also accelerated in combination-treated, compared with single drug-treated and DMSO-treated Ba/F3-ITD, MV4-11, and MOLM-14 cells, and accelerated turnover was abrogated in cells pretreated with MG-132 (Fig. 4D), consistent with our previous results (12).

Pim and FLT3 Inhibitor Cotreatment Downregulates c-Myc Through Phosphorylation at T58
To test the role of c-Myc T58 phosphorylation in c-Myc downregulation by gilteritinib and Pim inhibitor cotreatment, Ba/F3-ITD cells infected with    inhibitor cotreatment, and the effect of overexpression of Myc with T58A was due to expression of a non-phosphorylatable variant rather than to a general effect of Myc overexpression.

FLT3 and Pim Inhibitor Cotreatment Downregulates Mcl-1 Through Phosphorylation at S159
To Apoptosis induction by gilteritinib and AZD1208 combination was also significantly reduced in cells infected with pBabe-Flag hMcl-1-S159A compared with pBabe-Flag hMcl-1 plasmid or pBabe-puro empty vector, while apoptosis induction by single drugs did not differ (Fig. 6C).Thus Mcl-1 S159 phosphorylation contributes to apoptosis induction by gilteritinib and AZD1208 combination.

Gilteritinib and Pim Inhibitor Combination Rapidly Inactivates AKT, but AKT Inactivation is not Necessary for GSK-3β Activation, c-Myc or Mcl-1 Downregulation or Apoptosis Induction
We recently showed that PP2A-activating drugs enhance FLT3 inhibitor efficacy in cells with FLT3-ITD through AKT inhibition, which activates GSK-3β, resulting in GSK-3β-mediated enhanced c-Myc and Pim-1 proteasomal degradation (24).We therefore studied the possible role of AKT inhibition in posttranslational c-Myc and Mcl-1 downregulation in cells with FLT3-ITD treated with gilteritinib and Pim inhibitor combination.
To test whether AKT inactivation is necessary for GSK-3β activation and c-Myc

ERK1/2 is Inactivated Similarly by Gilteritinib with or without AZD1208
To determine differential effects of gilteritinib and Pim inhibitor treatment, relative to gilteritinib treatment, on ERK1/2 inactivation, Ba/F3-ITD cells were treated with gilteritinib and/or AZD1208, or DMSO control, and expression of p-ERK and ERK was measured at serial timepoints by immunoblotting.Both gilteritinib and AZD1208 and gilteritinib alone similarly rapidly inactivated ERK1/2 (Supplementary Fig. S2D).

Discussion
FLT3-ITD is present in blasts of 30% of patients with AML and is associated with poor treatment outcomes.FLT3 inhibitors have clinical activity, but efficacy is generally limited and transient.We previously showed that concurrent targeting of the serine/threonine kinase Pim-1, which is upregulated downstream of STAT5 in the aberrant FLT3-ITD signaling pathway (6, 7), enhances apoptosis induction by FLT3 inhibitors by posttranslationally downregulating the antiapoptotic protein Mcl-1 through enhanced proteasomal degradation (12).Here we show that concurrent targeting of Pim-1 and FLT3 in cells with FLT3-ITD also causes posttranslational downregulation of c-Myc, preceding    We found that posttranslational downregulation of both c-Myc and Mcl-1 resulted from activation of GSK-3β, which phosphorylates these proteins at T58 and S159, respectively, thereby tagging them for proteasomal degradation.We previously found that PP2A-activating drugs, which overcome inactivation of the tumor suppressor PP2A in cells with FLT3-ITD, also enhance efficacy of FLT3 inhibitors through activation of GSK-3β, which phosphorylates c-Myc and Pim-1, increasing posttranslational downregulation of both proteins and enhancing apoptosis (24).We did not study Mcl-1, but it is likely also downregulated.
FLT3 and Pim inhibitor cotreatment was not AKT-dependent or ERKdependent, and enhanced efficacy thus likely occurred entirely through the STAT5 pathway.GSK-3 is a substrate of AKT in the (PI3K)-Akt-mTOR pathway (21).In our work here, gilteritinib and Pim inhibitor cotreatment inhibited AKT inhibition and activated GSK-3β, but GSK-3β activation was independent of AKT inhibition, as it occurred in cells with (constitutively activated) myr-AKT.
Moreover, AZD1208 and gilteritinib cotreatment downregulated c-Myc and Mcl-1 and induced apoptosis similarly in cells infected with myr-AKT and with empty vector.In contrast, we previously showed that PP2A-activating drug and FLT3 inhibitor cotreatment rapidly inactivated AKT in FLT3-ITD-expressing cells through dephosphorylation at both S473 and T308, and that AKT inactivation caused GSK-3β activation.In addition, PP2A-activating drug and FLT3 inhibitor cotreatment caused GSK-3β activation in parental Ba/F3-ITD cells and Ba/F3-ITD cells infected with empty vector, but not with myr-AKT, and treatment with the AKT inhibitor MK-2206 caused GSK-3β activation (24).
GSK-3β activation has also been reported to be ERK dependent (46), but ERK was similarly inactivated by gilteritinib and AZD1208 and by gilteritinib alone.
As above, enhanced efficacy of Pim and FLT3 inhibitor treatment thus likely occurred entirely through enhanced effects on the STAT5 pathway (9).
The two GSK-3 paralogs, GSK-3α and GSK-3β, have variably been reported to have tumor suppressor and oncogenic properties in different acute leukemia types and subtypes (47,48).GSK-3α or GSK-3β was found to be necessary for survival and proliferation of cells with KMTA (MLL) gene rearrangements in vitro and in vivo, through destabilization of p27 Kip1 , a cyclin-dependent kinase inhibitor that is a tumor suppressor protein (49).Treatment with selective GSK-3 inhibitors, knockdown of GSK-3α or genetic ablation of GSK-3β inhibited proliferation of cells with KMTA rearrangements, and this antiproliferative effect was inhibited by p27Kip1 knockdown (49).In contrast, GSK-3 inhibition did not produce antiproliferative effects and did not increase expression of p27Kip1 in acute leukemia cells with other gene rearrangements, including TEL-AML, EA-HLF, and EA-PBX (49).Of note, GSK-3 inhibitors have also exhibited clinical activity in some patients with refractory solid tumors, and the roles of GSK-3 and consequences of its modulation would seem to be contextual and tumor specific (50).
Prognostic significance of both GSK-3 phosphorylation and localization has also been reported in AML.(54).
In chronic myeloid leukemia (CML), which is driven by the oncogenic tyrosine kinase BCR-ABL, GSK-3 was reported to be inactivated and enforced expression of constitutively active GSK-3 reduced proliferation and potentiated BCR-ABL inhibitor-induced apoptosis in both BCR-ABL inhibitor-sensitive and -resistant cells (55).This work suggested therapeutic efficacy of GSK-3 activation in CML.Indeed, the Pim-1 kinase inhibitor SMI-4a was subsequently found to exert antitumor effects in both imatinib-sensitive and -resistant CML cells by increasing GSK-3β activity (56).
GSK-3β activation has also been described in response of colorectal cancer cells to kinase inhibitors.In one study, treatment with the multi-kinase inhibitor sorafenib inactivated ERK1/2 by dephosphorylation at T202/Y204, resulting in GSK-3β activation by dephosphorylation at S9, promoting p65 phosphorylation and expression of the proapoptotic Bcl-2 protein family member PUMA (57).In a subsequent study, treatment of colorectal cancer cells with gilteritinib, which inhibits AXL as well as FLT3, caused AKT inhibition and consequent GSK-3β activation, which resulted in nuclear translocation of p65 and induction of PUMA as a mechanism of apoptosis induction, and GSK-3β knockdown suppressed gilteritinib-induced p65 phosphorylation and induction of PUMA (58).The same mechanism was demonstrated for the multi-kinase inhibitor regorafenib in colorectal cancer cells (59).
On the basis of our work here and our previous work (24), GSK-3β activation appears to be an effective strategy for optimizing response to FLT3 inhibitors, through posttranslational downregulation of c-Myc, Mcl-1, and Pim-1, key proteins driving proliferation and resistance to apoptosis in AML cells with FLT3-ITD.This pathway also appears to be relevant to other tyrosine kinase-driven leukemias, as well as solid tumors.
Finally, our data provide support for potential development of a clinical trial combining TP-3654 and gilteritinib in AML with FLT3-ITD, with correlative laboratory studies.Gilteritinib is FDA approved for treatment of relapsed and refractory FLT3-mutated AML (29).AZD1208 was generally well tolerated in a phase I trial in patients with AML and solid tumors in two dose-escalation studies, but it increased CYP3A4 activity after multiple dosing, resulting in increased drug clearance, and was therefore withdrawn from clinical studies (60).
determine the effects of gilteritinib and Pim inhibitor cotreatment on expression of c-Myc, relative to Mcl-1, Ba/F3-ITD, MV4-11, and MOLM-14 cells and blasts from a patient with AML with FLT3-ITD were treated with gilteritinib and/or AZD1208, or DMSO control, and Ba/F3-ITD, MV4-11, and MOLM-14 cells were also treated with gilteritinib and/or TP-3654, or DMSO control.Samples collected at serial timepoints were analyzed by immunoblotting.c-Myc was downregulated to a greater extent by combination treatment than by gilteritinib alone, as was Mcl-1, and c-Myc downregulation preceded Mcl-1 downregulation

FIGURE 2
FIGURE 2 TP-3654 enhances efficacy of gilteritinib in vivo.NRG mice injected intravenously with MV4-11-luc cells were treated with gilteritinib and/or TP-3654, or vehicle control, with first treatment day defined as day 1.A, Serial images of mice in first experiment, treated with gilteritinib 7.5 mg/kg and/or TP-3654 50 mg/kg, or vehicle control, once every other day for three doses, followed by 2 days rest, each week.B, Changes in photon intensity, measured by bioluminescence imaging, over time, with P = 0.0043, comparing gilteritinib and TP-3654 combination versus gilteritinib alone on day 60 by two-way ANOVA with Sidak multiple comparison test.C, Survival curves, with P = 0.0027 by Kaplan-Meier analysis.Median survival was 51, 44, 51, and 65 days for mice treated with vehicle, TP-3654, gilteritinib, and gilteritinib and TP-3654 combination, respectively.D, Serial images of mice in second experiment, treated with gilteritinib 15 mg/kg and/or TP-3654 50 mg/kg, or vehicle control, daily for 5 days each week.E, Change in photon intensity, measured by bioluminescence imaging, over time, with P < 0.0001, comparing gilteritinib and TP-3654 combination versus gilteritinib alone by day 35 by two-way ANOVA.

FIGURE 4 c
FIGURE 4 c-Myc and Mcl-1 are downregulated through enhanced proteasomal degradation.A and B, Ba/F3-ITD, MV4-11, and MOLM-14 cells plated at 1 × 10 5 cells/mL were treated with 100 μg/mL CHX for 1 hour to inhibit protein synthesis, with or without addition of the proteasome inhibitor MG-132 after 30 minutes, prior to treatment with gilteritinib and/or AZD1208 at same concentrations as in Fig. 3, or DMSO control.Samples collected at serial timepoints were studied for expression of c-Myc (A), Mcl-1 (B) and vinculin loading control protein by immunoblotting.Data in A and B are shown graphically and numerically in C and D, respectively.
determine whether c-Myc and Mcl-1 downregulation is caused by GSK-3β activation, Ba/F3-ITD and MV4-11 cells and FLT3-ITD AML patient blasts were treated with gilteritinib and AZD1208 in the presence and absence of the GSK-3β inhibitor TG-C 24, and c-Myc and Mcl-1 expression was measured by immunoblotting at serial timepoints.TC-G 24 treatment inhibited c-Myc and Mcl-1 downregulation by gilteritinib and AZD1208 cotreatment in Ba/F3-ITD and MV4-11 cells and FLT3-ITD AML primary patient blasts (Fig. 7C and D), demonstrating that c-Myc and Mc-1 downregulation by cotreatment results at least in part from GSK-3β activation.

and Mcl- 1
downregulation, Ba/F3-ITD cells infected with myr-AKT plasmid, which renders AKT constitutively active, or empty vector control were treated with AZD1208 and/or gilteritinib, or DMSO control.AZD1208 and gilteritinib cotreatment activated GSK3-β and downregulated c-Myc and Mcl-1 (Supplementary Fig.S2B), and induced apoptosis (Supplementary Fig.S2C) similarly in cells infected with myr-AKT or empty vector control.Therefore, while Pim and FLT3 inhibitor combination rapidly inactivates AKT, AKT inactivation is not necessary for GSK3-β activation, c-Myc or Mcl-1 downregulation or apoptosis induction by gilteritinib and AZD1208 combination treatment.

FIGURE 6
FIGURE 6 S159A-mutated Mcl-1 confers resistance to Mcl-1 downregulation and to apoptosis induction by gilteritinib and AZD1208 combination treatment.A, Ba/F3-ITD cells infected with pBabe-Flag hMcl-1-S159A, containing Mcl-1 with a mutation changing serine to alanine at residue 159, preventing phosphorylation, pBabe-Flag hMcl-1 plasmid, containing wild-type Mcl-1, or pBABE-puro empty vector were treated with either gilteritinib and AZD1208 or DMSO control, and serial samples were immunoblotted for Mcl-1 and vinculin loading control.Densitometric analysis is also shown.B, To measure Mcl-1 protein turnover, cells were pretreated with CHX for 1 hour and then treated with gilteritinib and AZD1208 (+) or DMSO control (−).Serial samples were immunoblotted for c-Myc and vinculin loading control.Densitometric analysis was performed.Mcl-1 was normalized to vinculin and 50% protein turnover timepoints were determined to be 1.75 versus more than 2 hours for gilteritinib and AZD1208 versus DMSO control for empty vector, 1.27 versus 1.6 for wild-type Mcl-1 and more than 2 hours for both for S159A Mcl-1.C, Cells infected with pBabe-Flag hMcl-1-S159A, pBabe-Flag hMcl-1 or pBABE-puro empty vector control were treated with gilteritinib and/or AZD1208, or DMSO control, for 48 hours, and apoptosis was measured.Apoptosis induction by gilteritinib and AZD1208 combination was significantly reduced in cells infected with pBABE-puroS159A compared with empty vector control (**, P < 0.001).

FIGURE 8
FIGURE 8 Summary figure.Summary figure showing the proposed effects of combined FLT3 and Pim kinase inhibition in cells with FLT3-ITD.
and remains in active clinical development.As an additional consideration, because Pim and FLT3 inhibitor combination treatment enhances Mcl-1 downregulation, relative to FLT3 inhibitor alone, and Mcl-1 upregulation is a mechanism of resistance to the Bcl-2 inhibitor venetoclax(61), in vitro, in vivo and potentially clinical exploration of TP-3654 with giletritinib and venetoclax(62,36) as triple combination therapy would be of interest.