IN VIVO ACTIVITY OF THE SECOND-GENERATION PROTEASOME INHIBITOR IXAZOMIB AGAINST PEDIATRIC T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA XENOGRAFTS

Abstract

The overall survival rate of patients with T-cell acute lymphoblastic leukemia (T-ALL) is now 90%, although patients with relapsed T-ALL face poor prognosis. The ubiquitin-proteasome system maintains normal protein homeostasis, and aberrations in this pathway are associated with T-ALL. Here we demonstrate the in vitro and in vivo activity of ixazomib, a second-generation orally available, reversible, and selective proteasome inhibitor against pediatric T-ALL cell lines and patient-derived xenografts (PDXs) grown orthotopically in immune-deficient NSG mice. Ixazomib was highly potent in vitro, with IC₅₀ values in the low nanomolar range. As a monotherapy, ixazomib significantly extended mouse event-free survival of five out of eight T-ALL PDXs in vivo.

INTRODUCTION

While overall survival rates for pediatric acute lymphoblastic leukemia (ALL) are approximately 90%, patients with relapsed T-cell ALL (T-ALL) have a particularly poor prognosis.¹ Therefore, there is an urgent need to develop novel therapies that target relapsed/refractory ALL. The ubiquitin-proteasome system (UPS) maintains normal protein homeostasis and is responsible for a host of cellular processes including intracellular protein degradation, apoptosis, inflammation, and cell cycle control.² Dysregulation of UPS-controlled processes has been linked to many cancers, rendering the UPS an attractive therapeutic target leading to the development of proteasome inhibitors.³

Clinical validation of proteasome inhibitors has been demonstrated for bortezomib, a first-in-class proteasome inhibitor that obtained US Food and Drug Administration (FDA) approval for the treatment of multiple myeloma (MM) and mantle cell lymphoma in 2003.^4,5 Although bortezomib exhibited limited activity against pediatric leukemia as a single agent,⁶ phase I and II combination studies elicited complete remission (CR) rates of 67–73%.^7,8 However, a phase III study of bortezomib added to induction therapy and to the delayed intensification treatment block failed to show benefit for children with newly diagnosed T-ALL.⁹

The next generation proteasome inhibitor carfilzomib obtained FDA approval for MM in 2012⁵ and exhibited increased specificity, potency, and cellular apoptotic sensitivity compared to bortezomib in preclinical ALL models.^10,11 A phase I study investigating the efficacy of carfilzomib in combination with standard chemotherapy in newly-diagnosed adults with ALL elicited a 100% CR rate and minimal residual disease negativity in 80% of patients.¹⁰

Ixazomib is a second-generation orally available, reversible and selective inhibitor of the β5 site of the constitutive proteasome, and the β1i and β5i sites of the immunoproteasome.^12–14 Preclinical studies demonstrated improved pharmacokinetic, pharmacodynamic, and antitumor properties of ixazomib over bortezomib, including a shorter proteasome-dissociation half-life from red blood cells, which may lead to greater biodistribution.^12,15 In 2015, ixazomib received FDA approval for use in combination with lenalidomide and dexamethasone in MM.¹⁶ Given that leukemia cells are more susceptible to apoptosis following proteasome inhibition compared to healthy cells,^2,17 ixazomib is a promising candidate for preclinical ALL studies. Here we report the activity of ixazomib as a single agent against a panel of pediatric T-ALL patient-derived xenografts (PDXs).

METHODS

In vitro assessment of drug activity

Cytotoxicity assays were performed as described previously.^18,19 Briefly, Jurkat cells (American Type Culture Collection, Manassas, VA) were suspended in RPMI media supplemented with 10% fetal calf serum (Biosera, Shanghai, China) while PDX cells were cultured in QBSF-60 medium (Quality Biological Inc., Gaithersberg, MD) supplemented with Flt-3 ligand (20 ng/mL, BioNovus Life Sciences, Cherrybrook, NSW, Australia). Cells were plated according to the optimal seeding density (0.2–4 × 10⁵ cells/well) and PDX cells were incubated for 3 hours to equilibrate (37°C, 5% CO₂). Jurkat cells and PDX cells were treated with ixazomib at 10-fold dilutions (1 pM - 10 μM) or vehicle control (at the equivalent DMSO concentration) for 48 h, with the final concentration of DMSO being <0.1%. Viability was determined using the AlamarBlue reduction assay and read on a Victor X3 Multilabel Plate Reader (560 nm excitation and 590 nm emission; PerkinElmer, Waltham, MA). All readings were calculated with 0 h background subtraction and normalized as a percentage of control wells, and the half-maximal inhibitory concentration (IC₅₀) was calculated by interpolation of nonlinear regression curves calculated by GraphPad Prism 9 software. Full characterization of Jurkat cells and T-ALL PDX samples is provided in Table 1 and Supplementary Figure 1.

TABLE 1.

Patient demographics, key molecular lesions and in vivo ixazomib responses of the pediatric ALL PDXs used in the study

	Patient Demographics				PDX Molecular Characteristics				Ixazomib Response
PDX	ALL subtype	Age at diagnosis (years)	Sex	Disease status at biopsy	Structural variations	Copy number variations	Single nucleotide variants, gene (amino acid change, variant allele frequency)	TALL Sorts classification	EFST-C	EFST/C	Median ORM
ALL-8	T-ALL	12.6	M	Relapse	nd	CDKN 2A (HOM DEL), CDKN 2B (HOM DEL)	FAT2 (R2678*, 0.43); NT5C2 (R367Q, 0.56); FBXW7 (R465C, 0.42); ASXL1 (D863G, 0.47); SMARCA4 (R1189Q, 0.49); IL1B (R75K, 0.47); FBN2 (D1008H, 0.51)	TAL/LMO	2.8	1.24	PD1
ALL-16	T-ALL	10.2	F	Diag nosis	nd	nd	AURKB (V91M, 0.42); DYM (F573L, 0.44); BCL11B (C429fs, 0.26); ARID1A (1334_1335ins Q, 0.32); NOTCH1 (L1600P, 0.58); RPL10 (R98S, 0.65); PHLPP1 (S323P, 0.39)	NKX2	2.9	1.17	PD1
ALL-27	T-ALL	8.5	M	Diag nosis	nd	CDKN 2A (HOM DEL), CDKN 2B (HOM DEL)	ACTB (357_364ISKQ EYDE>M, 0.29); ACTB (E364fs, 0.39); NOTCH1 (L1678P, 0.51); CREBBP (S1934P, 0.52); FBXW7 (R479Q, 0.53); PIK3CD (C416R, 0.23)	TAL/LMO	1.1	1.21	PD1
ALL-30	T-ALL	7.4	M	Diag nosis	STIL::TAL	CDKN 2A (HOM DEL), CDKN 2B (DEL)	SETD2 (DGRES1788d el, 0.31); FLG (A2307T, 0.27); GLI2 (R1543C, 0.54); EYS (TDG985del, 0.36)	TAL/LMO	−1.0	0.91	PD1
ALL-31	T-ALL	10.1	M	Diag nosis	PTMA::NP M1; LMO1::TR BC1; LMO1::TR BC2	CDKN 2A (HOM DEL), CDKN 2B (HOM DEL)	NRG2 (A792T, 0.49); MLLT4 (R915H, 0.47)	TAL/LMO	1.9	1.17	PD1
ALL-32	T-ALL	11.1	M	Relapse	NUP214::A BL1	CDKN 2A (HOM DEL), CDKN 2B (HOM DEL)	ATF7IP (H115R, 0.48); ABL1 (E255K, 0.37); NOTCH2 (P2087T, 0.42); CTCF (, 0.45); NOTCH1 (L1593P, 0.47); WHSC1 (E1099K, 0.52); MTOR (A461V, 0.59); FGF3 (R145Q, 0.48)	TLX3	0.3	1.05	PD1
ETP-1	ETP	16	M	Diag nosis	ATRX::MI R325HG; TP53::SNX 31	nd	NOTCH1 (S2492*, 0.44); RUNX1 (A122fs, 0.47); NUP214 (A1376fs, 0.39); PHF6 (N147fs, 0.92); FGFR2 (E586Q, 0.43); ZFHX3 (G3511S, 0.51); EZH2 (S695L, 0.97); MED12 (R516H, 1); ZFP36L2 (A348T, 0.48); GNB1 (D76G, 0.51)	Diverse	3.6	1.26	PD1
ETP-3	ETP	14	M	Diag nosis	ERG::IGJ	CDKN 2A (HOM DEL), CDKN 2B (HOM DEL)	GATA3 (TSTPLW270f s, 0.27); GATA3 (STPLW271fs, 0.32); NOTCH1 (PEQ1582del, 0.34); NOTCH1 (P1582fs, 0.52); NRXN1 (L607W, 0.51); FBN2 (I1248V, 0.49); NOTCH1 (Q1584fs, 0.5); TERT (A67P, 0.34); NOTCH1 (P1582T, 0.57); GATA3 (N286T, 0.69); JAK1 (S703I, 1); SOS1 (R982Q, 0.38); GIGYF2 (R407Q, 0.38); NOTCH4 (T684M, 0.45)	TAL/LMO	0.4	1.03	PD1

DEL, shallow deletion; del, deletion; EFS, event-free survival; ETP, early T-cell precursor; fs, frameshift mutation; HOMDEL, homozygous deletion; ins, insertional mutation; Median ORM, median objective response measure; nd, not detected; T-C, treated-control; T/C, treated/control

^*,nonsense mutation.

Development of PDXs, drug treatments and assessment of in vivo drug activity

All animal experiments were approved by the Animal Care and Ethics Committee of UNSW Sydney (Sydney, Australia). Experiments used continuous PDXs previously established in 20–25 g female non-obese diabetic/severe combined immuno-deficient (NOD.CB17-Prkdc^scid/SzJ, NOD/SCID) or NOD/SCID/interleukin-2 receptor γ–negative (NOD.Cg-Prkdc^scidIL2rg^tm1Wjl/SzJAusb, NSG) mice, as described previously.^19–21 Briefly, leukemia cells were inoculated intravenously into 6–8 week-old NOD/SCID or NSG mice (Australian BioResources, Moss Vale, NSW, Australia) and leukemic burden was monitored by enumeration of human CD45⁺ (%huCD45⁺) cells versus total CD45⁺ leukocytes (human plus murine) in the peripheral blood (PB) and tissues, as described previously. ^19–21 Details of the in vivo Ixazomib citrate treatment including objective response measures used to assess event-free survival (EFS) is provided in supplementary methods.

RESULTS & DISCUSSION

Previous research using T-ALL and acute myeloid leukemia (AML) cell lines showed that bortezomib and ixazomib preferentially and reversibly inhibited the β5 subunit of the constitutive proteasome, and the equivalent β5i subunit of the immunoproteasome, although the activity of bortezomib was more potent.¹³ Conversely, ixazomib elicited greater β1 and β1i inhibition, even in bortezomib-resistant cell lines, indicating that the mechanism of action of ixazomib, while similar to bortezomib, is not identical.¹³ Therefore, we first assessed the efficacy of ixazomib using in vitro cytotoxicity assays against a T-ALL cell line (Jurkat) and two T-ALL PDXs (ALL-30, ALL-31, Table 1). Ixazomib elicited IC₅₀ values in the low nM range (Jurkat, 1.9 nM; ALL-30, 1.6 nM; ALL-31, 2.8 nM; Supplementary Figure 2), demonstrating excellent in vitro potency of ixazomib in cell lines and more clinically relevant T-ALL PDXs.

We previously reported that bortezomib induced regressions in two B- and two T-ALL PDXs and significantly extended mouse event-free survival (EFS).²² The bortezomib data, along with its demonstrated activity in relapsed/refractory pediatric ALL,^7,8 provided strong rationale for the testing of additional proteasome inhibitors against T-lineage PDXs. Prior to in vivo efficacy testing, we first evaluated the tolerability of ixazomib in both naïve and leukemia engrafted NSG mice

In the naïve cohort (n=18), ixazomib was generally well tolerated with only one case of toxicity observed at each of the 3 mg/kg and 4.5 mg/kg doses after the second treatment. Although some weight loss was observed throughout the treatment, all remaining mice recovered (Supplementary Figure A.3). Biochemistry (Supplementary Table 1) and hematology (Supplementary Table 2) analyses fell mostly within the normal range and were not suggestive of acute toxicity. In mice engrafted with a T-ALL PDX, ALL-31 (Supplementary Figure B.3 and C.3; n=24), ixazomib was poorly tolerated at the two highest doses, with severe toxicity observed after the first dose (Supplementary Figure D.3). Ixazomib was better tolerated at the two lower doses (1.5 and 3 mg/kg), and no mice were euthanized due to toxicity. Biochemistry and hematology analyses revealed no drug-related perturbations (Supplementary Table 3 and Supplementary Table 4). These findings necessitated a dose reduction of ixazomib to 3 mg/kg for the efficacy study which was below the company recommended maximum tolerated dose (MTD).¹³

Using this revised dose, the efficacy of ixazomib as a monotherapy was evaluated in vivo against six T-ALL PDXs and two early T-cell precursor ALL PDXs (ETP-ALL; Table 1) which have previously been molecularly annotated.²³ Ixazomib significantly delayed disease progression in five out of eight PDXs, with Treated-Control (T-C) values ranging from −1.0 to 3.6 days and T/C values from 0.91 to 1.26 (Figures 1 and 2, Table 1). Each PDX achieved a score of Progressive Disease 1 (PD1) according to stringent objective response measures.²³ (Supplementary Table 5).

FIGURE 1.

In vivo activity of ixazomib against pediatric ALL PDXs. Individual mouse %huCD45⁺ cells in peripheral blood (left panels) and Kaplan–Meier curves for EFS (right panels) for T-ALL PDXs ALL-8, ALL-16, ALL-27, ALL-30. Vehicle controls: gray lines; drug treated: orange lines. In engraftment plots, individual mice are represented by pale colored lines, while the median value for the cohort is shown as a dark line. The dashed horizontal line at 25% indicates the threshold for event. (PD1 = progressive disease 1 as part of the objective response criteria; EFS = event free survival; T-C = the difference in median time-to-event (days) between Treatment and Control groups)

FIGURE 2.

In vivo activity of ixazomib against pediatric ALL PDXs. Individual mouse %huCD45⁺ cells in peripheral blood (left panels) and Kaplan–Meier curves for EFS (right panels) for T-ALL PDXs ALL-31 and ALL-32 and ETP-ALL PDXs ETP-1 and ETP-3. Vehicle controls: gray lines; drug treated: orange lines. In engraftment plots, individual mice are represented by pale colored lines, while the median value for the cohort is shown as a dark line. The dashed horizontal line at 25% indicates the threshold for event. (PD1 = progressive disease 1 as part of the objective response criteria; EFS = event free survival; T-C = the difference in median time-to-event (days) between Treatment and Control groups)

To date, ixazomib has been evaluated in combination with standard-of-care agents in several hematological malignancies. Ixazomib has been evaluated in multiple phase I/II/III trials for both newly-diagnosed and relapsed/refractory MM, and has FDA approval for use in combination with lenalidomide and dexamethasone for the treatment of patients with MM who have received at least one prior therapy. The combination of ixazomib, cyclophosphamide and dexamethasone elicited high overall response rates in newly-diagnosed MM.^24–26 Ixazomib has also been evaluated in combination regimens for patients with AML and ALL, although the contribution of ixazomib to the activity observed for these regimens was not isolated in these clinical trials.^27,28 Ixazomib is currently being evaluated in a phase I/II clinical trial in combination with chemotherapy (vincristine, dexamethasone, asparaginase, doxorubicin [VXLD]) in children with relapsed/refractory ALL or lymphoblastic lymphoma (ClinicalTrials.gov Identifier NCT03817320). In adult ALL, ixazomib was evaluated in a Phase I trial in newly-diagnosed patients aged 51–75 who were treated with ixazomib in combination with prednisone, vincristine, and doxorubicin, with Philadelphia chromosome positive patients also treated with dasatinib.²⁸ Ixazomib was well tolerated and, despite the poor outcomes associated with adult-onset ALL, the combination therapy induced an overall remission rate of 79%.²⁸ Combination of bortezomib with VXLD in relapsed/refractory pediatric ALL in a phase I/II clinical trial resulted in an overall response rate of 72.9% with no significant difference observed in 2-year overall survival in B- versus T-lineage patients.²⁹ Additionally, bortezomib in combination with chemotherapy in relapse pediatric ALL exhibited a compete response rate of 68 ± 10% in T-ALL patients.³⁰ However, when bortezomib was added to standard therapy for children with newly diagnosed T-ALL in the Children’s Oncology Group AALL1231 clinical trial, there was no added benefit.⁹ Therefore, further evaluation of ixazomib in combination with standard of care therapies for T-ALL will require new biological insights to guide patient selection and the selection of agents for combination testing.

In summary, here we show that ixazomib has low nanomolar activity against a T-ALL cell line and T-ALL-PDX cells ex vivo and elicited modest in vivo activity as a monotherapy against pediatric T-ALL PDXs.

Highlights:

• Ixazomib is an orally available, reversible selective proteasome inhibitor

• Ixazomib elicited IC₅₀ values in the low nM range in T-ALL cell models in vitro

• Ixazomib treatment of PDX models in vivo resulted in a modest increase in survival