Phenotypic Drug Response Profiling Identifies Asparaginase-Based Synergistic Combinations for Very High Risk Acute Lymphoblastic Leukaemia
Authors: Sidhu Jasmeet , Steffen Fabio D. , Jimenez Iria Anne , Huang Yun , Rauwolf Kerstin K. , Chakraborty Arijit , Krishnan Shekhar , Saha Vaskar , Bornhauser Beat , Bourquin Jean-Pierre 
Published: December 2023

In acute lymphoblastic leukaemia (ALL), empirical multi-drug chemotherapy combinations lead to &gt;85% cure rates in children. But 10-15% of patients show suboptimal response and as drugs are dosed to tolerance, further intensification is not possible. An exception is the key antileukaemic drug, asparaginase (ASNase), which is not associated with dose-related toxicity. We evaluated the use of ex-vivo co-culture based drug response profiling (DRP) to identify novel synergistic and effective drug combinations with ASNase for patients with suboptimal responses to standard chemotherapy in ALL.

Patient-derived xenograft (PDX) ALL cells from 5 patients with high-risk B-ALL (3 B-other, 2 IKZF plus) were treated with 120 drugs in 5-point serial dilutions in combination with or without an IC 50 dose of ASNase, in co-cultures with hTERT-immortalized primary bone marrow stromal cells. Eight clinically relevant agents with promising sensitizing activity as identified by decreased area-under-the-curve with addition of ASNase were identified (SEL, selinexor; ELTA, eltanexor; VEN, venetoclax; BZM, bortezomib; navitoclax; carfilzomib; mitoxantrone, birinapant).

We validated combinations of ASNase with the 8 selected drugs plus 4 drugs used in induction therapy (prednisolone, dexamethasone, vincristine and daunorubicin) using a 4x4 drug matrix in 15 high-risk ALL PDX samples. Synergy scores were calculated using the zero-interaction potency (ZIP) model. Synergy (ZIP score ≥10) or additivity (ZIP score &gt;0 and &lt;10) with ASNase was identified for exportin-1 inhibitors (XPO1i; selinexor, eltanexor), venetoclax and proteosomal inhibitors (bortezomib, carfilzomib) in 100% (15/15), 86.6% (13/15) and 80-86.6% (12/15 and 13/15) samples respectively. Antagonism (ZIP score &lt;0) was noted in more than one-third (6/15) of samples with ASNase in combination with steroids or vincristine. Three-drug combinations were evaluated ex-vivo in these 15 PDX samples. Synergy was identified with addition of low doses of BZM or VEN to the ASNase-SEL combination (ASNase-SEL-BZM and ASNase-SEL-VEN).

Next, 4-6 mice per treatment arm were transplanted intravenously with 1 million ALL cells from 4 high-risk B-ALL patients. Randomized cohorts were treated with vehicle, ASNase-BZM-ELTA (ABE) and ASNase-dexamethasone-vincristine (ADV). Leukemic burden was assessed weekly using flowcytometry. The mice tolerated both treatment arms well. Platelet counts recovered within one week after an initial drop in the ABE arm. Both treatment groups had significant improvement in event free survival (EFS) as compared to the control group (ABE p-value 0.0082; ADV p-value 0.0325) though no significant differences were noted between the 2 treatment arms (p-value 0.8971).

Three patients with high-risk medullary relapsed/refractory BCP-ALL, 2 not in morphological remission and 1 at time of very early relapse, received a combination of ASNase-BZM-VEN-SEL (ABVS) after consent. One patient for whom DRP suggested sensitivity to both SEL and VEN achieved molecular remission (molecular minimal residual disease &lt; 10 -5) after one course of therapy remaining in complete remission 11 months later. Two patients with sensitivity to SEL but not to VEN as assessed by DRP, had progressive disease. ABVS was well tolerated in these patients with no grade 3-4 adverse events.

Our results identify XPO1 inhibitors as a new class of drugs with promising anti-leukaemic activity in BCP-ALL. Co-culture based DRP shows promise in identifying alternative novel sensitive synergistic combinations ex-vivo with decreased toxicity for patients who have an inadequate response to standard therapy.