Jacqueline S. Garcia

Fold change of all genes in the CRISPRi screen

Acute myeloid leukemia (AML) is prone to relapse driven by therapy-persistent residual cells. To discover specific vulnerabilities in this population, we performed genome-wide CRISPR interference screens in leukemia cells treated with multiple agents. KHSRP was the top hit, the depletion of which sensitized AML cells to therapy and substantially prolonged survival in treated AML-bearing mice. Analysis of in vivo residual disease after venetoclax/azacitidine treatment identified downregulation of the vitamin C and uric acid transporter SLC23A1, which mediated resistance to multiple therapies. KHSRP depletion restored SLC23A1 expression by preventing its ZC3H4-mediated nuclear mRNA degradation. KHSRP depletion therefore enhanced the synergistic cytotoxicity of vitamin C and uric acid, particularly in therapy-persistent leukemia cells. Re-expression of TET2 overrode the chemosensitizing effect of KHSRP depletion in TET2-mutant leukemia, suggesting that KHSRP-linked phenotypes were related to vitamin C– and uric acid–mediated TET activation. These findings suggest targeting KHSRP to enhance treatment efficacy and selectively eradicate residual AML. Significance:Residual AML cells that persist following therapy have distinct biological properties compared with initial disease or overt relapse. Here, we nominate KHSRP as a target to improve therapy efficacy and exploit selective vulnerabilities of AML residual disease.

Genes with differential splicing after KHSRP depletion

Supplementary Table S1 from Therapy-Persistent Leukemia Is Selectively Vulnerable to SLC23A1 Restoration via Targeting KHSRP

Figure S1. KHSRP depletion selectively reduces residual leukemia cell viability. Figure S2. SLC23A1 expression is reduced in residual leukemia populations. Figure S3. KHSRP promotes SLC23A1 mRNA decay via ZC3H4. Figure S4. KHSRP depletion reduces residual leukemia cell persistence after various chemotherapies.

Sequences of guide RNA and cDNA

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an orphan, aggressive hematologic malignancy characterized by high CD123 expression. Tagraxofusp (TAG), a CD123-directed toxin, is the first approved therapy. Prior studies demonstrated BPDCN dependence on BCL2, sensitivity to venetoclax (VEN), and reversal of TAG resistance with azacitidine (AZA). We conducted a phase 2 study evaluating combination therapy with TAG, AZA, and VEN in patients with BPDCN. Patients with previously untreated (1L) or relapsed/refractory (R/R) BPDCN received 28-day cycles of AZA (75 mg/m2 on days 1-7), VEN (400 mg on days 1-21), and TAG (12 μg/kg on days 4-6). Eligibility followed TAG guidelines to mitigate risk of capillary leak syndrome (CLS). Twenty-seven patients were enrolled (16 1L, 11 R/R), with median age of 70 years (range, 21-81). Composite complete remission (composed of: complete remission [CR], CR with incomplete count recovery, and CR clinical) rates were 88% in 1L and 64% in R/R cohorts. Median duration of response was not reached in 1L and was 7.2 months in R/R patients. CLS occurred in 15% of patients, most were grade 2. In the 1L cohort, median overall survival (OS) and progression-free survival (PFS) were not reached; the 2-year OS was 65% and 2-year PFS was 53%. Median OS in R/R patients was 8.4 months. A high proportion of patients proceeded to allogeneic stem cell transplantation in remission (63% in 1L, including 10/11 patients aged ≤75 years; 55% in R/R). TAG-AZA-VEN is highly active in both 1L and relapsed BPDCN with a predictable and manageable safety profile, supporting its use as a new therapeutic option. This trial was registered at www.clinicaltrials.gov as #NCT03113643.

Although adults with Philadelphia chromosome-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL) may achieve a measurable residual disease-negative (MRD-) complete response (CR) after frontline treatment and blinatumomab (blina) consolidation, survival outcomes are heterogeneous, and known prognostic factors are limited. We conducted a retrospective, multicenter study of 81 adults with Ph+ B-ALL in MRD- (<10-4) CR1 after blina consolidation and stratified them by pre-blina MRD status as determined by multiparametric flow cytometry (flow). Patients who were pre-blina flow MRD- (n = 56) and flow MRD+ (n = 25) had similar baseline characteristics. With a median follow-up of 31.0 months, pre-blina flow MRD- patients had superior disease-free (DFS) and overall survival (OS) on univariate (p = 0.049 and p = 0.045, respectively) and multivariable analyses (HR 0.27, 95% CI 0.08-0.87 and HR 0.17, 95% CI 0.03-0.90, respectively) relative to pre-blina flow MRD+ patients. Inferior DFS was seen with the IKZF1-plus genotype (HR 3.51, 95% CI 1.08-11.44) and extramedullary disease at diagnosis (HR 3.90, 95% CI 1.19-12.73). In exploratory analyses, pre-blina flow MRD+ status remained associated with inferior DFS (p = 0.0005) and OS (p < 0.0001) for patients who did not receive a subsequent hematopoietic cell transplant (HCT) in CR1, but not in patients who did (p = 0.55 and p = 0.99, respectively). In summary, flow MRD+ status before blina consolidation was associated with inferior survival in patients with Ph+ B-ALL in CR1 despite subsequent clearance, and consolidative HCT may reduce this prognostic effect.

In relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL), responses to the CD22 antibody-drug conjugate inotuzumab ozogamicin (INO) are frequent but short-lived. Based on preclinical evidence of synergy, we conducted a phase 1 trial of the BCL-2 inhibitor venetoclax (VEN) plus standard-dose INO for adults with R/R CD22+ ALL/LBL. Twenty-three patients enrolled (15 ALL, 8 LBL): three at dose level 1 (DL1; VEN 200 mg/day), six at DL2 (VEN 400 mg/day), and fourteen in an expansion cohort (VEN 400 mg/day). The recommended dose was VEN 400 mg/day for 21 days per cycle. No dose-limiting toxicities or early mortality occurred. Patients received a median of 2 cycles (range 1-5). The most common grade ³3 adverse events were thrombocytopenia (43.5%) and neutropenia (39.1%). Four patients (17.4%) developed sinusoidal obstructive syndrome. Of 22 evaluable patients, 21 (95.5%) achieved complete remission, including 19 after one cycle. Measurable residual disease (MRD) cleared in 16/18 (88.9%) by flow cytometry (<10-4) and 14/19 (73.7%) by next-generation sequencing (<10-6). Fourteen (63.6%) patients proceeded to HSCT. With a median follow-up of 25.3 months (95% CI 19.0-32.4), two-year disease-free (DFS) and overall survival (OS) were 48% (95% CI 24-72%); median DFS was 22.1 months (95% CI 10.4-NA), and median OS was not reached. MRD-positivity was associated with lower baseline BCL-2 dependence. Correlates of progression included acquired MCL-1 dependence, CD22 antigen escape, drug efflux gene ABCB1 expression, and oncogenic mutations. In summary, VEN+INO is safe and effective for R/R B-ALL/LBL, producing frequent and durable MRD-negative remissions. NCT05016947

Acute myeloid leukemia (AML) is prone to relapse driven by therapy-persistent residual cells. To discover specific vulnerabilities in this population, we performed genome-wide CRISPR interference screens in leukemia cells treated with multiple agents. KHSRP was the top hit, the depletion of which sensitized AML cells to therapy and substantially prolonged survival in treated AML-bearing mice. Analysis of in vivo residual disease after venetoclax/azacitidine treatment identified downregulation of the vitamin C and uric acid transporter SLC23A1, which mediated resistance to multiple therapies. KHSRP depletion restored SLC23A1 expression by preventing its ZC3H4-mediated nuclear mRNA degradation. KHSRP depletion therefore enhanced the synergistic cytotoxicity of vitamin C and uric acid, particularly in therapy-persistent leukemia cells. Re-expression of TET2 overrode the chemosensitizing effect of KHSRP depletion in TET2-mutant leukemia, suggesting that KHSRP-linked phenotypes were related to vitamin C– and uric acid–mediated TET activation. These findings suggest targeting KHSRP to enhance treatment efficacy and selectively eradicate residual AML. Significance: Residual AML cells that persist following therapy have distinct biological properties compared with initial disease or overt relapse. Here, we nominate KHSRP as a target to improve therapy efficacy and exploit selective vulnerabilities of AML residual disease.