New Targets – New therapies Hairy Cell Leukemia: New Targets – New therapies Robert J. Kreitman, M.D.

HAIRY CELL LEUKEMIA B-cell leukemia 2% of all leukemias, ~1000 cases/yr ‘Fried egg’ appearance Cytoplasmic projections CD19, CD22, CD103, CD11c, CD25

313 HCL randomized between DCF and IFN NEJM (1984) 310:15 3 CR, 4 PR (n=8) (other trials, CR 5-10%, PR 30-70%, DFS 1.5-3Y) NEJM (1987) 316:825 16 CR, 10 PR (n=27) (other trials: CR 70-75%, PR 3-25%, DFS: 8Y 76% JCO, 13:974, 1995 313 HCL randomized between DCF and IFN CR rates: DCF 76% vs IFN 11%, (p<0.001) DCF CR 66% in patients crossing over after IFN

Single 5-7 day course of treatment NEJM (1990) 322:1117 11/12 CR, 1/12 PR CR 75-87%, PR 11-25%, DFS: 4Y 79-84%, 7Y 60-66% Single 5-7 day course of treatment Evidence is lacking that cladribine can eradicate HCL: Saven et al., Blood 92:1918, 1998, Goodman et al, JCO 21:891, 2003 No plateau seen on DFS curves Sigal et al., Blood, 115:1893, 2010. Of 358 HCL, MRD was present in 10 of 19 patients still in CR 16y later.

Outcome of HCL after purine analog Else et al., BJH, 145:733, 2009 Median RFS 16y (1st line), 11y (2nd line), 6.5y (3rd line) Median time to purine refractory disease ~ 34 years Golomb et al, Ann Int Med, 89:677, 1978 – Median OS of HCL = 4 years

HCLc and HCLv Clinical features: Type HCLc HCLv % of HCL ~80-90% ~10-20% Spleen enlargement Mild Severe Lymph nodes - + Cytopenias + - Lymphocytosis - + CD25 + - CD103 + +/- Annexin A1 + - Response to CdA, DCF 70-87% CR 8% CR

Classic HCL HCLv

Classic HCL HCLv VH4-34+ HCL Arons et al., Blood, 14:4687, 2009

Mab-based treatment for HCL Blood (2003) 102:810 13% CR, 13% PR (n=24) All patients had prior purine analog and needed treatment Better results if no prior purine analog, or no cytopenias. Rituximab with cladribine or pentostatin better, but more toxic

Cladribine followed by Rituximab For Early HCL Rituximab followed Cladribine by 1 month 100% CR in 31 patients, MRD+ in 6 (21%) of 28 In HCLv, 2 of 5 had CRs lasting 12 and 35 mo Ravandi et al., Blood, 118:3818, 2011

Pentostatin Cladribine Benzimidazole Ring Nitrogen mustard Structure of bendamustine compared with other purine analogs NH2 N N N N N N Cl N N N Pentostatin Cladribine O O HOCH2 HOCH2 OH Cl OH Benzimidazole Ring N N Cl Nitrogen mustard COOH N Bendamustine CH3

C1D24 C7D42 Response in HCL adenopathy to bendamustine Kreitman et al., Leuk & Lymphoma, 52:1153, 2011

Burotto et al., CCR, in Press Bendamustine and rituximab for multiply relapsed/refractory HCL Cy1 Cy2 Cy3 Cy4 Cy5 Cy6 | | | | | | | | | | | | D1 D15 D1 D15 D1 D15 D1 D15 D1 D15 D1 D15 Bendamustine 70 mg/m2 d1,2 (n=6) Bendamustine 90 mg/m2 d1,2 (n=6+28) Rituximab 375 mg/m2 d1, 15 Bendamustine Dose (mg/m2 d1, 2) 70 90 Patients enrolled 6 6 ORR (CR+PR) 6 (100%) 6 (100% CR 3 (50%) 4 (67%) MRD-negative 2 (33%) 4 (67%) Med Time to CR 223 days 111 days Burotto et al., CCR, in Press

Recombinant immunotoxins for HCL Blood (1999) 94:3340 25% CR, 75% PR (n=4) (LMB-2) NEJM (2001) 345:241 69% CR, 13% PR (n=16) JCO (2005) 23:6719 61% CR, 19% PR (n=31) (BL22)

BL22 BL22

Moxetumomab pasudotox: High affinity mutant of BL22 >1000 CLL HCL BL22 V V V V L L L H Median ratio II Ib III 1000 2.4 (n=32) - S Moxe 100 V V V V L L L H II Ib III IC50 (ng/ml) - S 10 SSY  THW at 100a, b, c. Affinity  14x (lower koff). Originally HA22, renamed CAT-8015, then moxetumomab pasudotox. Salvatore et al, CCR, 8:995, 2002 1 Median ratio 8.3 (n=30) 0.1 BL22 Moxe BL22 Moxe

Moxetumomab pasudotox in HCL 7 CR 13 (46%) PR 11 (39%) 6 SD 3 (11%) PD 1 (4%) 5 CR 46%, ORR 86% (n=28) PATIENTS 4 12 (92%) of 13 CRs > 1yr 3 2 1 10 20 30 40 50 dose (ug/Kg x3) Kreitman et al., JCO, 30:1823,2012

NEJM, 364:2305, 2011 Total exome sequencing of 1 HCL patient BRAF V600E & in 47 other HCL patients vs 0 of 195 other hematologic tumors BRAF V600E also in 50% melanomas, 7% colon cancers, other cancers HCLv not characterized Multiple studies confirmed 100% expression of BRAF V600E in HCL BRAF WT in 16/16 HCLv, 5/5 classic HCL expressing VH4-34, & 6 others Thus, HCL is a heterogeneous disease with some variants defined only molecularly, and BRAF can be WT even in classic HCL MEK mutations have been found in HCLv and VH4-34+ HCL

Rapid development of agents targeting the MAPK pathway 6/30/11 (NEJM) Vemurafenib vs DTIC for V600E+ melanoma showed ^OS, PFS, leading to FDA approval for melanoma. Chapman et al., NEJM, 364:2507, 2011

Vemurafenib 240 mg q12 x56 days Dietrich S et al., NEJM, 366(21):2038-40, 2012

6-Month follow-up of HCL treated with Vemurafenib Dietrich S et al., JCO, 31:e300, 2013

Transient response of HCL to Vemurafenib (240 mg po q12 x58 days) Pre Day 37 Day 60 Follows et al., BJH, 161:136, 2013 Thus, Vemurafenib can induce response including CR in HCL, but durability and eradication of MRD has not been reported, and relapse occurs.

Other anecdotal responses of HCL to Vemurafenib 27y history of HCL, multiply relapsed (14 times). Began Vemurafenib 960 mg po q12hr x3 weeks Stopped due to arthralgias Arthralgias resolved HCL decreased by BMBx from 68% to <5% by morphology and 10-20% by IHC. 23y history of HCL, multiply relapsed Plt 24, Hgb 6.9 Began Vemurafenib 240 mg po q12hr x56d Day 56: Improved Plt to 253, Hgb 9.6 Day 56: BMBx improved to 5% HCL Day 90: CR achieved, but MRD not reported Munoz et al., JCO, 31:e351, 2013 Peyrade et al., Haematologica, 98:e20, 2013 Thus, Vemurafenib can induce response including CR in HCL, but durability and eradication of MRD has not been reported, and relapse occurs.

Rational for combined BRAF-MEK inhibition (preclinical) BRAFV600E human melanoma xenograft 500 1000 1500 2000 10 20 30 40 50 60 70 80 90 Untreated control Trametinib (0.3 mg/kg) (anti-MEK) Mean tumor volume (mm3) + SEM Dabrafenib (30 mg/kg) Dabrafenib (300 mg/kg) (anti-BRAF) Dabrafenib + trametinib (30/0.3 mg/kg) Treatment period (days) Control BRAFi (150 mg/kg) BRAFi + MEKi (150 + 1.5 mg/kg) BRAFi alone results in thickening & hyperkeratosis Co-dosing with MEKi attenuates lesion formation Conclusions: Besides more effective, combined treatment is less toxic than single-agent BRAF or MEK inhibition 24

Mutated BRAF no inhibition BRAF V600E inhibition MEK inhibition BRAF+ MEK inhibition B-Raf V600E B-Raf V600E B-Raf V600E B-Raf V600E Malignant phenotype SCC Kerato-acantoma Rash Diarrhea Lack of toxicity

Conclusions: Purine analogs cladribine and pentostatin are highly effective in 1st-2nd line for HCL, but are usually non-curative and MRD is very common Patients with HCLv or VH4-34+ classic HCL are primarily resistant Rituximab has limited activity in HCL as a single agent, but high activity combined with purine analogs Bendamustine or BR is active in relapsed/refractory HCL LMB-2 (anti-CD25) is active for CD25+ relapsed/refractory HCL Moxetumomab pasudotox (HA22), an affinity-matured version of BL22, achieves CRs in ~50% of relapsed/refractory HCL without DLT or MRD BRAF is an important target in most but not all HCL. Vemurafenib is a useful agent in V600E+ HCL but MRD-negative CR is unreported. New targets in HCL and HCLv are being sought for therapy.