Background and Significance:

TP53 mutant Acute Myeloid Leukemia (TP53m AML) defines the most treatment-resistant AML subtype with a median overall survival of 5 months and a 3% 5-year survival. Thus, despite significant effort heretofore, this subtype remains an unmet need.

Leukemia stem cells (LSCs) are the subset of AML cells responsible for evading therapy and persisting to cause recurrence. To define the signaling pathways that are activated in TP53m LSCs, we used CyTOF to profile human AML samples. TP53m LSCs displayed significantly elevated levels of activated NF kappa B (NFkB) and pSTAT1, demonstrating a unique signaling activation state in TP53m AML relative to other subtypes. Next, we asked whether the elevated NFkB levels in TP53m LSCs depend on mutant p53. Indeed, we found that knockdown of TP53 reduced NFkB protein levels in Kasumi AML cells, which are TP53m, but not in MOLM13 AML cells, which are TP53WT. RNA sequencing showed TP53 knockdown led to loss of NFkB and LSC self-renewal signatures in Kasumi cells. These data suggest TP53m LSCs depend on mutant p53-driven activation of NFkB for self-renewal. We then used proteasome inhibitors to test whether NFkB inhibition can kill TP53m AML. We treated TP53m AML samples with carfilzomib and bortezomib (BTZ). Each drug reduced NFkB levels and led to a significant reduction of serial colony formation. Next, we treated TP53 knockdown and control Kasumi cells with BTZ. In control cells, which harbor TP53m, BTZ reduced stem cell markers and activated signaling molecules. However, the effect of BTZ was significantly attenuated in TP53 knockdown cells, suggesting the effects of BTZ in AML are enhanced by mutant p53. Together, these data suggest that TP53m AML rely on the NFkB pathway for self-renewal and inhibiting this pathway with BTZ may target the LSCs of TP53m AML.

BTZ has previously been shown to synergize with chemotherapy. BTZ in combination with standard AML chemotherapy was previously shown to be to be safe and tolerable. However, this combination was not adapted for widespread clinical use because it did not significantly enhance the baseline complete remission rates of approximately 70% in unselected AML patients. Notably, the complete remission rates with standard chemotherapy are much lower in TP53m AML (34-51%). Since TP53mAML comprises a small subset of AML (8-13%), such patients were likely poorly represented in the prior BTZ trials. Our pre-clinical data suggest that TP53m patients would be particularly sensitive to proteasome inhibition; therefore, in this trial we combine CPX-351 and BTZ for upfront TP53m AML.

Study Design and Methods:

This is a single center, phase I/II study evaluating the safety and efficacy of BTZ with CPX-351 in newly diagnosed TP53m AML. Induction consists of BTZ administered on days 1, 4, 8, and 11 in combination with CPX-351 on day 1, 3, and 5. Response assessment is performed at count recovery but no later than day 35. Subsequent therapy will be determined by the treating physician. Blood and marrow samples for correlative objectives will be collected prior to treatment initiation and at each subsequent bone marrow biopsy as well as blood following the first and fourth dose of BTZ. Patients treated at MTD in phase I will be included in Phase II. Expected enrollment is 12 patients for phase I and 22 patients in phase II.

The primary endpoint of phase I is to define safety and tolerability and preliminary efficacy in phase II. Phase I will determine the MTD of BTZ in combination with CPX-351 with 4 total BTZ dose levels via the continuous reassessment method with a DLT toxicity proportion of 20% or less. Phase II's primary efficacy measure is CR rate. Secondary outcomes are ORR, MRD status after induction, CR rate at any time within the first year, time to relapse, and 1-year and 2-year OS. Correlative objectives include AML burden, LSC frequency, self-renewal transcription profiles, and immune cell subset in the TME.

Participants must be adults who have newly diagnosed AML based on ELN 2022 classification with TP53 gene mutation documented either by p53 overexpression by IHC or by next generation sequencing considered pathological or likely pathological using the OncoKD database. APL and patients with extramedullary or CNS disease are excluded. The protocol is under IND 175618, and the first patient is expected to be enrolled in August 2025.

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2025
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